Multispecific polypeptide constructs having constrained cd3 binding and related methods and uses

ABSTRACT

The invention relates generally to multispecific polypeptides having constrained CD3 binding. In some embodiments, components of the multispecific polypeptides are connected by a non-cleavable linker. Also provided are methods of making and using these multispecific polypeptides in a variety of therapeutic, diagnostic and prophylactic indications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application62/656,331, filed Apr. 11, 2018, entitled “MULTISPECIFIC POLYPEPTIDECONSTRUCTS HAVING CONSTRAINED CD3 BINDING AND RELATED METHODS AND USES,”the contents of which are incorporated by reference in their entiretyfor all purposes.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled744952000200SeqList.TXT, created Apr. 10, 2019 which is 212 kilobytes insize. The information in the electronic format of the Sequence Listingis incorporated by reference in its entirety.

FIELD

The invention relates generally to multispecific polypeptides havingconstrained CD3 binding. In some embodiments, components of themultispecific polypeptides are connected by a non-cleavable linker. Alsoprovided are methods of making and using these multispecificpolypeptides in a variety of therapeutic, diagnostic and prophylacticindications.

BACKGROUND

Therapeutic antibodies that cause target cell depletion generally relyon effector functions mediated via interaction with Fc-gamma-receptors(FcγRs) and complement proteins. Effector cells expressing FcγRs arepredominately those of the innate immune system. T-cells are not directeffector cells involved in antibody mediated target cell depletion.

CD3 (Cluster of Differentiation 3) T-cell co-receptor is a multimericprotein composed of four distinct polypeptide chains, referred to as theε, γ, δ, and ζ chains. The CD3 complex serves as the signaling module ofthe T cell receptor that associates non-covalently with theantigen-binding a/b chains of T cell receptor (TCR).

Because direct engagement of CD3 results in T-cell activation, it is adesirable target for a variety of therapeutic and/or diagnosticindications. Accordingly, there exists a need for antibodies andtherapeutics that target the CD3/TCR pathway.

SUMMARY

The present disclosure provides multispecific polypeptide constructsthat exhibit constrained CD3 binding. In some embodiments, themultispecific polypeptide construct is composed of a first componentcomprising an immunoglobulin Fc region and a second component comprisinga CD3-binding region, wherein the first and second components arecoupled by a linker, such as a non-cleavable linker, wherein the Fcregion is positioned N-terminal to the CD3-binding region; and one orboth of the first and second components comprises an antigen bindingdomain that binds a tumor associated antigen (TAA). In some embodiments,the CD3-binding region binds CD3 (CD3ε). In some embodiments, theantigen binding domain is positioned amino-terminally relative to the Fcregion and/or carboxy-terminally relative to the CD3 binding region ofthe multispecific polypeptide construct. In some embodiments, the firstcomponent comprises a first antigen binding domain and the secondcomponent comprises a second antigen binding domain, wherein each of theantigen binding domains bind a tumor associated antigen (TAA). In somecases, the first antigen binding domain is positioned at the aminoterminus of the multispecific construct and the second antigen bindingdomain is positioned at the carboxy terminus of the multispecificconstruct. In some embodiments, the first antigen binding domain ispositioned amino-terminally relative to the Fc region and/orcarboxy-terminally relative to the CD3 binding region of themultispecific polypeptide construct. In particular embodiments ofprovided multispecific polypeptide constructs, at least one antigenbinding domain is positioned carboxy-terminally relative to the CD3binding region of the multispecific polypeptide construct.

Provided herein is a multispecific polypeptide construct, wherein themultispecific construct comprises in order, from N-terminus toC-terminus: a first antigen binding domain that binds to atumor-associated antigen (TAA); an immunoglobulin Fc region; a linker,such as a non-cleavable linker, a CD3 binding region that binds CD3(CD3ε); and a second antigen binding domain that binds atumor-associated antigen (TAA). Also provided is a multispecificpolypeptide construct, wherein the multispecific construct comprises inorder, from N-terminus to C-terminus: an immunoglobulin Fc region; alinker, such as a non-cleavable linker; a CD3 binding region that bindsCD3 (CD3ε); and an antigen binding domain that binds a tumor-associatedantigen (TAA). Provided is a multispecific polypeptide construct,wherein the multispecific construct comprises in order, from N-terminusto C-terminus: an antigen binding domain that binds to atumor-associated antigen (TAA); an immunoglobulin Fc region; a linker,such as a non-cleavable linker; and a CD3 binding region that binds CD3(CD3ε).

In some of any of the provided embodiments, the linker is anon-cleavable linker. In some embodiments, the linker is a linker thatdoes not contain a substrate recognition site specific to cleavage by aprotease.

In some of any of the provided embodiments, the positioning of the Fcregion N-terminal to the CD3 binding region reduces or prevents theability of the CD3 binding region to bind CD3. In some embodiments, thefirst component (component #1) and the second component (component #2)of the multispecific polypeptide constructs are linked and binding toCD3 is disallowed, unless the antigen binding domain(s) is bound to itscognate antigen. In some embodiments, component #1 contains at least oneantigen binding domain and an Fc region. In some embodiments, component#2 contains at least a CD3 binding region domain and an antigen bindingdomain, the former of which is capable of binding CD3 (when themultispecific construct is bound to antigen recognized by the antigenbinding domain or domains of component #1 or component #2). Thus,linkage of the CD3 binding region to the Fc region as described ensuresthat the multispecific polypeptide constructs do not bind or otherwiseengage CD3 unless the antigen binding domain(s) is bound to its cognateantigen. This is advantageous as it prevents systemic binding of the CD3binding region to T-cells and instead focuses the CD3 binding region'sability to bind to site of antigen expression. This is beneficial as itdiminishes or eliminates a major binding sink of peripheral T-cells,potentially allowing more favorable distribution and localization atsite of antigen expression, e.g., tumor cells or the tumormicroenvironment.

When the antigen binding domain(s) is bound to its cognate antigen, themultispecific polypeptide construct, via component #2, is capable offorming an immune synapse between an antigen-expressing cell and aT-cell. This co-engagement mediates antigen dependent T-cell activation,cytotoxicity, cytokine release, degranulation and proliferation. In someembodiments, the multispecific polypeptide constructs are capable ofinteracting with FcγRs and mediating innate immune effector functions,for example antibody dependent cellular cytotoxicity (ADCC) and antibodydependent cellular phagocytosis (ADCP). In some embodiments, themultispecific polypeptide constructs are capable of interacting withcomplement proteins, namely C1q, and mediating complement-dependentcytotoxicity.

In some embodiments, the cognate antigen recognized by the antigenbinding domain(s) of a provided multispecific polypeptide construct is atumor associated antigen (TAA).

Thus, among the provided embodiments, the multispecific polypeptideconstruct is composed of a first component comprising an immunoglobulinFc region and a second component comprising a CD3-binding region,wherein the first and second components are coupled by a linker, such asa non-cleavable linker, wherein the Fc region is positioned N-terminalto the CD3-binding region; and one or both of the first and secondcomponents comprises an antigen binding domain that binds a tumorassociated antigen (TAA). In some embodiments, the CD3-binding regionbinds CD3 (CD3ε). In some embodiments, the antigen binding domain ispositioned amino-terminally relative to the Fc region and/orcarboxy-terminally relative to the CD3 binding region of themultispecific polypeptide construct. In some embodiments, the firstcomponent comprises a first antigen binding domain and the secondcomponent comprises a second antigen binding domain, wherein each of theantigen binding domains bind a tumor associated antigen (TAA). In somecases, the first antigen binding domain is positioned at the aminoterminus of the multispecific construct and the second antigen bindingdomain is positioned at the carboxy terminus of the multispecificconstruct. In some embodiments, the first antigen binding domain ispositioned amino-terminally relative to the Fc region and/orcarboxy-terminally relative to the CD3 binding region of themultispecific polypeptide construct. In particular embodiments ofprovided multispecific polypeptide constructs, at least one antigenbinding domain is positioned carboxy-terminally relative to the CD3binding region of the multispecific polypeptide construct.

In some embodiments, the CD3 binding region is an antibody or an antigenbinding fragment. In particular embodiments, the antibody or antigenbinding fragment is a two chain polypeptide containing a variable heavy(VH) and a variable light (VL) chain. In some embodiments, the antibodyor antigen-binding fragment is an Fv. In particular embodiments, the Fvis a disulfide-stabilized Fv (dsFv) containing an interchain disulfidebond between the VH and VL chains.

Provided herein is a multispecific polypeptide construct comprising afirst component comprising an immunoglobulin Fc region and a secondcomponent comprising a CD3-binding region, wherein the CD3 bindingregion is an anti-CD3 antibody or antigen binding fragment that is an Fvantibody fragment comprising a variable heavy chain region (VH) and avariable light chain region (VL); the Fc is a heterodimeric Fccomprising a first Fc polypeptide and a second Fc polypeptide and the VHand VL of the anti-CD3 antibody or antigen binding fragment are linkedto opposite polypeptides of the heterodimeric Fc; the first and secondcomponents are coupled by a non-cleavable linker, wherein the Fc regionis positioned N-terminal to the CD3-binding region; and the firstcomponent comprises a first antigen binding domain and the secondcomponent comprises a second antigen binding domain, wherein each of theantigen binding domains bind a tumor associated antigen (TAA). In someembodiments, the CD3-binding region binds CD3 (CD3ε). In someembodiments, the first antigen binding domain is positionedamino-terminally relative to the Fc region of the multispecificconstruct and the second antigen binding domain is positionedcarboxy-terminally relative to the CD3 binding region of themultispecific construct. In some embodiments, the multispecificconstruct comprises in order, from N-terminus to C-terminus: the firstantigen binding domain that binds to a tumor-associated antigen (TAA);the immunoglobulin Fc region; the non-cleavable linker; the CD3 bindingregion, wherein the CD3 binding region binds CD3 (CD3ε); and the secondantigen binding domain that binds a tumor-associated antigen (TAA).

Provided herein is a multispecific polypeptide construct comprising afirst component comprising an immunoglobulin Fc region and a secondcomponent comprising a CD3-binding region, wherein: the CD3 bindingregion is an anti-CD3 antibody or antigen binding fragment that is adisulfide-stabilized Fv antibody fragment (dsFv) comprising a variableheavy chain (VH) and a variable light chain (VL); the Fc is aheterodimeric Fc comprising a first Fc polypeptide and a second Fcpolypeptide and the VH and VL of the anti-CD3 antibody or antigenbinding fragment are linked to opposite polypeptides of theheterodimeric Fc; the first and second components are coupled by anon-cleavable linker, wherein the Fc region is positioned N-terminal tothe CD3-binding region; and one or both of the first and secondcomponents comprises an antigen binding domain that binds a tumorassociated antigen (TAA). In particular embodiments of providedmultispecific polypeptide constructs, at least one antigen bindingdomain is positioned carboxy-terminally relative to the CD3 bindingregion of the multispecific polypeptide construct. In some embodiments,the CD3-binding region binds CD3 (CD3ε).

Provided herein is a multispecific polypeptide construct comprising afirst component comprising an immunoglobulin Fc region and a secondcomponent comprising a CD3-binding region, wherein: the CD3 bindingregion is an anti-CD3 antibody or antigen binding fragment that is a anFv antibody fragment comprising a variable heavy chain (VH) and avariable light chain (VL); the Fc is a heterodimeric Fc comprising afirst Fc polypeptide and a second Fc polypeptide and the VH and VL ofthe anti-CD3 antibody or antigen binding fragment are linked to oppositepolypeptides of the heterodimeric Fc; the first and second componentsare coupled by a non-cleavable linker, wherein the Fc region ispositioned N-terminal to the CD3-binding region; and one or both of thefirst and second components comprises an antigen binding domain thatbinds a tumor associated antigen (TAA), wherein the antigen-bindingdomain is a single chain antibody fragment, such as an sdAb or an scFv.In particular embodiments of provided multispecific polypeptideconstructs, at least one antigen binding domain is positionedcarboxy-terminally relative to the CD3 binding region of themultispecific polypeptide construct. In some embodiments, theCD3-binding region binds CD3 (CD3ε).

In embodiments provided herein, the multispecific construct comprises inorder, from N-terminus to C-terminus: the first antigen binding domainthat binds to a tumor-associated antigen (TAA); the immunoglobulin Fcregion; the non-cleavable linker; the CD3 binding region, wherein theCD3 binding region binds CD3 (CD3ε); and the second antigen bindingdomain that binds a tumor-associated antigen (TAA).

In embodiments provided herein, the multispecific construct comprises inorder, from N-terminus to C-terminus: the immunoglobulin Fc region; thenon-cleavable linker; the CD3 binding region, wherein the CD3 bindingregion binds CD3 (CD3ε); and an antigen binding domain that binds atumor-associated antigen (TAA).

In embodiments provided herein, the multispecific construct comprises inorder, from N-terminus to C-terminus: the antigen binding domain thatbinds to a tumor-associated antigen (TAA); the immunoglobulin Fc region;the non-cleavable linker; and the CD3 binding region, wherein the CD3binding region binds CD3 (CD3ε).

Provided herein is a multispecific polypeptide construct, wherein themultispecific construct comprises in order, from N-terminus toC-terminus: a first antigen binding domain that binds to atumor-associated antigen (TAA); an immunoglobulin Fc region; a linker,such as a non-cleavable linker; a CD3 binding region that binds CD3(CD3ε); and a second antigen binding domain that binds atumor-associated antigen (TAA). Also provided is a multispecificpolypeptide construct, wherein the multispecific construct comprises inorder, from N-terminus to C-terminus: an immunoglobulin Fc region; alinker, such as a non-cleavable linker; a CD3 binding region that bindsCD3 (CD3ε); and an antigen binding domain that binds a tumor-associatedantigen (TAA). Provided is a multispecific polypeptide construct,wherein the multispecific construct comprises in order, from N-terminusto C-terminus: an antigen binding domain that binds to atumor-associated antigen (TAA); an immunoglobulin Fc region; a linker,such as a non-cleavable linker; and a CD3 binding region that binds CD3(CD3ε).

In some aspects, the antigen binding domain, or independently each ofthe antigen binding domains, is selected from an antibody or antigenbinding fragment, a natural cognate binding partner, an Anticalin(engineered lipocalin), a Darpin, a Fynomer, a Centyrin (engineeredfibroneticin III domain), a cystine-knot domain, an Affilin, anAffibody, or an engineered CH3 domain. In some embodiments, the naturalcognate binding partner comprises an extracellular domain or bindingfragment thereof of the native cognate binding partner of the TAA, or avariant thereof that exhibits binding activity to the TAA.

In some embodiments, the antigen-binding domain(s) includes one or morecopies of an antibody or an antigen-binding fragment thereof. In someembodiments, the antigen-binding domain(s) includes one or more copiesof an antibody or an antigen-binding fragment thereof selected from thegroup consisting of a Fab fragment, a F(ab′)₂ fragment, an Fv fragment,a scFv, a scAb, a dAb, a single domain heavy chain antibody, and asingle domain light chain antibody. In some embodiments, theantigen-binding domain(s) include one or more copies of one or moresingle domain antibody (sdAb) fragments, for example V_(H)H, V_(NAR),engineered V_(H) or V_(K) domains. V_(H)Hs can be generated from camelidheavy chain only antibodies. V_(NAR)s can be generated fromcartilaginous fish heavy chain only antibodies. Various methods havebeen implemented to generate monomeric sdAbs from conventionallyheterodimeric V_(H) and V_(K) domains, including interface engineeringand selection of specific germline families.

In some embodiments, the one or more antigen binding domainsindependently bind an antigen that is a tumor associated antigen (TAA).In some examples, the antigen binding domain, or independently each ofthe antigen binding domains, binds to a tumor antigen selected fromamong 1-92-LFA-3, 5T4, Alpha-4 integrin, Alpha-V integrin, alpha4beta1integrin, alpha4beta7 integrin, AGR2, Anti-Lewis-Y, Apelin J receptor,APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9,(Lewis a), Carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20,CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44,CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81,CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138,CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), CLAUDIN-3,CLAUDIN-4, cMet, Collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10,CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1,EDA, EDB, EGFR, EGFRviii, Endothelin B receptor (ETBR), ENPP3, EpCAM,EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2,FGFR3, FGFR4, FLT-3, Folate receptor alpha (FRa), GAL3ST1, G-CSF,G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GP IIb/IIIa receptors,Gp130, GPIIB/IIIA, GPNMB, GRP78, HER2/neu, HER3, HER4, HGF, hGH, HVEM,Hyaluronidase, ICOS, IFNalpha, IFNbeta, IFNgamma, IgE, IgE Receptor(FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R,IL-12Rbeta1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R,IL27/IL27R (wsx1), IL29, IL-31R, IL31/IL31R, IL2R, IL4, IL4R, IL6, IL6R,Insulin Receptor, Jagged Ligands, Jagged 1, Jagged 2, KISS1-R, LAG-3,LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, Mesothelin,MRP4, MUC1, Mucin-16 (MUC16, CA-125), Na/K ATPase, NGF, Nicastrin, NotchReceptors, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, OX-40, PAR2,PDGF-AA, PDGF-BB, PDGFRalpha, PDGFRbeta, PD-1, PD-L1, PD-L2,Phosphatidyl-serine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4,Sphingosine 1 Phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFbeta,TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFalpha,TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, Transferrin, Transferrin receptor,TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D,VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3.

In some embodiments, the Fc region is a homodimeric Fc region. In someembodiments, the immunoglobulin Fc region of the first component is anIgG isotype selected from the group consisting of IgG1 isotype, IgG2isotype, IgG3 isotype, and IgG4 subclass. In some examples, the Fcregion is an Fc region of a human IgG1, a human IgG2, a human IgG3, or ahuman IgG4, or is an immunologically active fragment thereof. In someembodiments, the Fc region comprises a polypeptide comprises the aminoacid sequence set forth in SEQ ID NO: 1 or a sequence of amino acidsthat has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%sequence identity to SEQ ID NO:1. In some cases, the Fc region comprisesa polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2 or a sequence of amino acids that has at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:2. In someof any such embodiments, the Fc region comprises a polypeptide comprisesthe amino acid sequence set forth in SEQ ID NO: 4 or a sequence of aminoacids that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%sequence identity to SEQ ID NO:4. In some examples, the Fc regioncomprises a polypeptide comprises the amino acid sequence set forth inSEQ ID NO: 5 or a sequence of amino acids that has at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:5.In some examples, the Fc region comprises a polypeptide comprises theamino acid sequence set forth in SEQ ID NO: 6 or a sequence of aminoacids that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%sequence identity to SEQ ID NO:6.

In some embodiments, the immunoglobulin Fc region is a polypeptidecomprising an amino acid sequence that is derived from an amino acidsequence selected from the group consisting of SEQ ID NOs: 1-6.

In some embodiments, the immunoglobulin Fc region is a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 1-6. In some embodiments, the immunoglobulin Fc region is apolypeptide comprising an amino acid sequence that is at least 50%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1-6.

In some embodiments, the Fc region is a heterodimeric Fc region.

In some embodiments, the Fc region is a heterodimer containing a firstFc polypeptide and a second Fc polypeptide wherein one or both of thefirst and second Fc polypeptides of the heterodimeric Fc region are avariant Fc polypeptide comprising at least one modification to induceheterodimerization compared to an Fc region of human IgG1, human IgG2 orhuman IgG4. In some embodiments, the at least one modification is in orcompared to an Fc region of human IgG1. In some embodiments, the atleast one modification is in or compared to the Fc polypeptide set forthin SEQ ID NO:1 or an immunologically active fragment thereof. In somecases, one or both Fc polypeptides of the heterodimeric Fc regioncomprises at least one modification to induce heterodimerizationcompared to a polypeptide of a homodimeric Fc region, optionallycompared to the Fc polypeptide set forth in SEQ ID NO:1 or animmunologically active fragment thereof. In some embodiments, each ofthe Fc polypeptides of the heterodimeric Fc independently comprise atleast one amino acid modification. In some cases, the at least onemodification is selected from a steric modification(s), a knob-into-holemodification(s), a charge mutation(s) to increase electrostaticcomplementarity of the polypeptides, a modification(s) to alter theisoelectric point (p1 variant), or combinations thereof.

In some examples, the amino acid modification is a charge mutation toincrease electrostatic complementarity of the polypeptides. In someembodiments, the first and/or second Fc polypeptides comprise amodification in complementary positions, wherein the modification isreplacement with an amino acid having an opposite charge to thecomplementary amino acid of the other polypeptide. In some embodiments,the first or second polypeptide comprise a modification in complementarypositions, wherein the modification is replacement with an amino acidhaving an opposite charge to the complementary amino acid of the otherpolypeptide. In some embodiments, at least the first or second Fcpolypeptides each comprise a modification in complementary positions,wherein the modification is replacement with an amino acid having anopposite charge to the complementary amino acid of the otherpolypeptide. In some embodiments, the first and second Fc polypeptideseach comprise a modification in complementary positions, wherein themodification is replacement with an amino acid having an opposite chargeto the complementary amino acid of the other polypeptide.

In some examples, the amino acid modification is a knob-into-holemodification.

In some embodiments, the first Fc polypeptide of the heterodimeric Fccomprises the modification selected from among Thr366Ser, Leu368Ala,Tyr407Val, and combinations thereof and the second Fc polypeptide of theheterodimeric Fc comprises the modification T366W. In some cases, thefirst and second Fc polypeptides further comprise a modification of anon-cysteine residue to a cysteine residue, wherein the modification ofthe first polypeptide is at one of a position Ser354 and Y349 and themodification of the second Fc polypeptide is at the other of theposition Ser354 and Y349. In some embodiments, the first Fc polypeptidecomprises the modifications T366W/S354C and the second Fc polypeptidecomprises the modifications T366S/L368A/Y407V/Y349C. In someembodiments, the first Fc polypeptide comprises the modificationsL368D/K370S and the second Fc polypeptide comprises the modificationsS364K/E357Q.

In some embodiments, the first Fc polypeptide comprises themodifications L368D/K370S and the second Fc polypeptide comprises themodifications S364K/E357Q.

In some embodiments, at least one of the first and second polypeptidecomprises the modifications Q295E/N384D/Q418E/N421D.

In some embodiments, one of the first or second Fc polypeptide of theheterodimeric Fc further comprises a modification at residue Ile253. Insome instances, the modification is Ile253Arg. In some embodiments, oneof the first or second Fc polypeptide of the heterodimeric Fc furthercomprises a modification at residue His435. In some instances, themodification is His435Arg.

In some embodiments, the Fc region, such as the first and/or second Fcpolypeptide comprises a polypeptide that lacks Lys447.

In some of any of the provided embodiments, the first polypeptide of theheterodimeric Fc comprises the sequence of amino acids set forth in anyof SEQ ID NOS:82, 86 or 201, and the second polypeptide of theheterodimeric Fc comprises the sequence of amino acids set forth in anyof SEQ ID NOS:83, 87, 90, 92, 202 or 205. In some embodiments, the firstFc polypeptide and the second Fc polypeptide comprises sequencesselected from the group consisting of SEQ ID NOS: 82 and 83,respectively; SEQ ID NOS: 86 and 87, respectively; SEQ ID NOS: 201 and202, respectively; SEQ ID NOS: 82 and 90, respectively; SEQ ID NOS: 86and 92, respectively; and SEQ ID NOS: 201 and 205, respectively.

In some embodiments, the immunoglobulin Fc region is a polypeptidecomprising an amino acid sequence that is derived from an amino acidsequence selected from the group consisting of SEQ ID NOs: 1-6comprising one or modifications. In some embodiments, the immunoglobulinFc region is a polypeptide comprising an amino acid sequence that isderived from an amino acid sequence selected from the group consistingof SEQ ID NOs: 1-6 comprising one or modifications to preventglycosylation, to alter Fc receptor interactions, to reduce Fc receptorbinding, to enhance the interaction with CD32A, to reduce the complementprotein C1q binding, to extend the half-life, to enhance FcRn binding,to alter antibody-dependent cellular cytotoxicity (ADCC) and/orcomplement-dependent cytotoxicity (CDC), to induce heterodimerization,to prevent dimerization, to stabilize the homodimerization at theCH3:CH3 interface, and combinations thereof.

In some embodiments, modifications within the Fc region reduce bindingto Fc-receptor-gamma receptors while having minimal impact on binding tothe neonatal Fc receptor (FcRn). In some embodiments, the mutated ormodified Fc polypeptide includes the following mutations: Met252Tyr andMet428Leu or Met252Tyr and Met428Val (M252Y, M428L, or M252Y, M428V)using the Kabat numbering system.

In some embodiments, the Fc region comprises a polypeptide comprising atleast one modification to enhance FcRn binding. In some examples, themodification is at a position selected from the group consisting ofMet252, Ser254, Thr256, Met428, Asn434, and combinations thereof. Insome cases, the modification is at a position selected from the groupconsisting of Met252Y, Ser254T, Thr256E, Met428L, Met428V, Asn434S, andcombinations thereof. In some particular embodiments, the modificationis at position Met252 and at position Met428. In some cases, themodification is Met252Y and Met428L. In some cases, the modification isMet252Y and Met428V.

In some embodiments, the first polypeptide of the heterodimeric Fccomprises the sequence of amino acids set forth in any of SEQ ID NOS:94, 96 or 207, and the second polypeptide of the heterodimeric Fccomprises the sequence of amino acids set forth in any of SEQ ID NOS:98, 100, or 209. In some embodiments, the first Fc polypeptide and thesecond Fc polypeptide comprises sequences selected from the groupconsisting of SEQ ID NOS: 94 and 98, respectively; SEQ ID NOS: 96 and100, respectively; and SEQ ID NOS: 207 and 209, respectively.

In some embodiments, the Fc region comprises a polypeptide comprising atleast one modification to enhance FcγR binding. In some cases, themodification is modification at Ser239 or Ile332. In some embodiments,the glycosylation of the Fc region is modified to enhance FcγR bindingas compared to an unmodified Fc region. In some examples, the Fc regionlacks or has reduced fucose content.

In some embodiments, the Fc region comprises a polypeptide comprising atleast one amino acid modification that reduces effector function and/orreduces binding to an effector molecule selected from an Fc gammareceptor or C1q. In some embodiments, the one or more amino acidmodification is deletion of one or more of Glu233, Leu234 or Leu235.

In some embodiments, the first polypeptide of the heterodimeric Fccomprises the sequence of amino acids set forth in any of SEQ ID NOS:82,86, 94 or 96, and the second polypeptide of the heterodimeric Fccomprises the sequence of amino acids set forth in any of SEQ ID NOS:83,87, 90, 92, 98 or 100. In some embodiments, the Fc region comprises apolypeptide comprising at least one amino acid modification that reduceseffector function and/or reduces binding to an effector moleculeselected from an Fc gamma receptor or C1q. In some examples, the one ormore amino acid modification is deletion of one or more of Glu233,Leu234 or Leu235. In some aspects, the first polypeptide of theheterodimeric Fc comprises the sequence of amino acids set forth in anyof SEQ ID NOS: 84, 88, 95 or 97 and the second polypeptide of theheterodimeric Fc comprises the sequence of amino acids set forth in anyof SEQ ID NOS: 85, 89, 91, 93, 99 or 101.

In some embodiments, the first polypeptide of the heterodimeric Fccomprises the sequence of amino acids set forth in any of SEQ ID NOS:84, 88, 95, 97, 203 or 208 and the second polypeptide of theheterodimeric Fc comprises the sequence of amino acids set forth in anyof SEQ ID NOS: 85, 89, 91, 93, 99, 101, 204, 206 or 210. In someembodiments, the first Fc polypeptide and the second Fc polypeptidecomprises sequences selected from the group consisting of SEQ ID NOS: 84and 85, respectively; SEQ ID NOS: 88 and 89, respectively; SEQ ID NOS:203 and 204, respectively; SEQ ID NOS: 95 and 99, respectively; SEQ IDNOS: 97 and 101, respectively; SEQ ID NOS: 208 and 210, respectively;SEQ ID NOS: 84 and 91, respectively; SEQ ID NOS: 88 and 93,respectively; and SEQ ID NOS: 203 and 206, respectively.

In some embodiments, the CD3 binding region is an anti-CD3 antibody orantigen-binding fragment. In some embodiments, the anti-CD3 antibody orantigen binding fragment comprises a variable heavy chain region (VH)and a variable light chain region (VL). In some of any such embodiments,the CD3 binding region is monovalent.

In some embodiments, the anti-CD3 antibody or antigen binding fragmentis not a single chain antibody, optionally is not a single chainvariable fragment (scFv). In some embodiments, the Fc is a heterodimericFc and the VH and VL that comprise the anti-CD3 antibody or antigenbinding fragment are linked to opposite polypeptides of theheterodimeric Fc.

In some embodiments, the CD3 binding region is not able to, or is notsubstantially able to, bind or engage CD3 unless at least one of theantigen binding domains is bound to its TAA. In some aspects, the CD3binding region is not able to, or is not substantially able, to bind orengage CD3 unless at least two of the antigen binding domains is boundto their TAA(s).

In some embodiments, the multispecific polypeptide construct contains alinker that is a polypeptide linker. In some embodiments, the linker isa polypeptide of up to 25 amino acids in length. In some cases, thelinker is a polypeptide of from or from about 2 to 24 amino acids, 2 to20 amino acids, 2 to 18 amino acids, 2 to 14 amino acids, 2 to 12 aminoacids, 2 to 10 amino acids, 2 to 8 amino acids, 2 to 6 amino acids, 6 to24 amino acids, 6 to 20 amino acids, 6 to 18 amino acids, 6 to 14 aminoacids, 6 to 12 amino acids, 6 to 10 amino acids, 6 to 8 amino acids, 8to 24 amino acids, 8 to 20 amino acids, 8 to 18 amino acids, 8 to 14amino acids, 8 to 12 amino acids, 8 to 10 amino acids, 10 to 24 aminoacids, 10 to 20 amino acids, 10 to 18 amino acids, 10 to 14 amino acids,10 to 12 amino acids, 12 to 24 amino acids, 12 to 20 amino acids, 12 to18 amino acids, 12 to 14 amino acids, 14 to 24 amino acids, 14 to 20amino acids, 14 to 18 amino acids, 18 to 24 amino acids, 18 to 20 aminoacids or 20 to 24 amino acids. In some embodiments, the linker is apolypeptide that is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19 or 20 amino acids in length.

In some embodiments, the linker is 3 to 18 amino acids in length. Insome embodiments, the linker is 12 to 18 amino acids in length. In someembodiments, the linker is 15 to 18 amino acids in length. In someembodiments, the linker is a polypeptide that is 18 amino acids inlength.

In some embodiments, the non-cleavable linker does not contain asubstrate recognition site that is specifically recognized for cleavageby a protease. In some embodiments, the protease is produced by animmune effector cell, by a tumor, or by cells present in the tumormicroenvironment. In some embodiments, the protease is produced by animmune effector cell and the immune effector cell is an activated Tcell, a natural killer (NK) cell, or an NK T cell. In some embodiments,the protease is selected from among matriptase, a matrix metalloprotease(MMP), granzyme B, and combinations thereof. In some embodiments, theprotease is granzyme B.

In some embodiments, the linker comprises the amino acid sequence GS,GGS, GGGGS (SEQ ID NO:149), GGGGGS (SEQ ID NO:135) and combinationsthereof. In some embodiments, the linker comprises the amino acidsequence (GGS)n, wherein n is 1 to 10. In some embodiments, the linkercomprises the amino acid sequence (GGGGS)n (SEQ ID NO: 173), wherein nis 1 to 10. In some embodiments, the linker comprises (GGGGGS)n (SEQ IDNO:172), wherein n is 1 to 4.

In some embodiments, the linker is or comprises GGS. In someembodiments, the linker is or comprises GGGGS (SEQ ID NO: 149). In someembodiments, the linker is or comprises GGGGGS (SEQ ID NO: 135). In someembodiments, the linker is or comprises GGSGGS (“(GGS)₂”) (SEQ ID NO:10). In some embodiments, the linker is or comprises GGSGGSGGS(“(GGS)₃”) (SEQ ID NO: 11). In some embodiments, the linker is orcomprises GGSGGSGGSGGS (“(GGS)₄”) (SEQ ID NO: 12). In some embodiments,the linker is or comprises GGSGGSGGSGGSGGS (“(GGS)₅”) (SEQ ID NO: 13).In some embodiments, the linker is or comprises GGGGGSGGGGGSGGGGGS (SEQID NO: 119). In some embodiments, the linker is or comprisesGGSGGGGSGGGGSGGGGS (SEQ ID NO: 147). In some embodiments, the linker isor comprises GGGGSGGGGSGGGGS (SEQ ID NO:170). In some embodiments, thelinker is or comprises GGGGG (SEQ ID NO:192).

In some embodiments, the antigen binding domain and the immunoglobulinFc region of the first component (which in some cases is the firstantigen binding domain) are operably linked via one or more furtheramino acid linkers (referred to herein as an intra-component linker).The intra-component peptide linker of the first component (also calledLP1) can be a peptide linker such as any as described in Section 11.3.The intra-component linker present in the first component, i.e. linkingthe Fc region and an antigen binding domain, can be of various lengths,for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20amino acids in length. In some embodiments, these intra-componentlinkers are composed predominately of the amino acids Glycine andSerine, denoted as GS-linkers herein. In some embodiments, the GS-linkercomprises an amino acid sequence selected from the group consisting ofGGSGGS, i.e., (GGS)₂ (SEQ ID NO: 10); GGSGGSGGS, i.e., (GGS)₃ (SEQ IDNO: 11); GGSGGSGGSGGS, i.e., (GGS)₄ (SEQ ID NO: 12); andGGSGGSGGSGGSGGS, i.e., (GGS)₅ (SEQ ID NO: 13).

In some embodiments, the multispecific polypeptide construct comprisesat least (i) a first polypeptide comprising the first Fc polypeptide ofthe heterodimeric Fc region, the linker and the VH domain of theanti-CD3 antibody or antigen binding fragment thereof; and (ii) a secondpolypeptide comprising the second Fc polypeptide of the heterodimeric Fcregion, the linker and the VL domain of the anti-CD3 antibody or antigenbinding fragment thereof, wherein one or both of the first and secondpolypeptide comprise at least one antigen-binding domain that binds to atumor associated antigen (TAA). In some embodiments, the VH of theanti-CD3 antibody or antigen-binding fragment is on the same polypeptideas the at least one antigen-binding domain that binds to a tumorassociated antigen (TAA). In some embodiments, the polypeptidecomprising the VL of the anti-CD3 antibody or antigen-binding fragmentdoes not contain the at least one antigen-binding domain that binds to atumor associated antigen (TAA). In particular embodiments of providedmultispecific polypeptide constructs, at least one antigen bindingdomain is positioned carboxy-terminally relative to the CD3 bindingregion of the multispecific polypeptide construct.

In some embodiments, the second component includes one or more copies ofthe CD3 binding region.

In some embodiments, the CD3 binding region is an anti-CD3 antibody orantigen-binding fragment that includes one or more copies of an antibodyor an antigen-binding fragment thereof that is able to bind or engageCD3, such as CD3ε. In some embodiments, the anti-CD3 binding domainincludes one or more copies of an antibody or an antigen-bindingfragment thereof selected from the group consisting of a Fab fragment, aF(ab′)₂ fragment, an Fv fragment, a scFv, a scAb, a dAb, a single domainheavy chain antibody, and a single domain light chain antibody.

In some embodiments, the anti-CD3 binding domain includes an Fv antibodyfragment that binds CD3ε (referred to herein as an anti-CD3ε Fvfragment).

In some embodiments, the anti-CD3ε Fv antibody fragment includes anamino acid sequence selected from the group of SEQ ID NO: 32-81, 191,196-200, 211, and 212. In some embodiments, the anti-CD3ε Fv antibodyfragment includes an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acidsequence selected from the group consisting of SEQ ID NO: 32-81, 191,196-200, 211, and 212. In some embodiments, the anti-CD3ε Fv antibodyfragment includes a combination of an amino acid sequence selected fromthe group of SEQ ID NO: 32-62, 196-198, and 211 and an amino acidsequence selected from the group consisting of SEQ ID NO: 63-81, 191,199, 200, and 212. In some embodiments, the anti-CD3ε Fv antibodyfragment includes a combination of an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto an amino acid sequence selected from the group consisting of SEQ IDNO: 32-62, 196-198, and 211 and an amino acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to anamino acid sequence selected from the group consisting of SEQ ID NO:63-81, 191, 199, 200, and 212.

In some embodiments, the anti-CD3ε Fv antibody fragment is a disulfidestabilized anti-CD3 binding Fv fragment (dsFv).

In some embodiments, the first component includes one or more copies ofan antigen-binding domain. In certain embodiments, the first componentcontains at least two antigen binding domains, such as two antigenbinding domains. In some embodiments, the at least two antigen bindingdomains of the first component bind to the same TAA. In some cases, theat least two antigen binding domains of the first component bind todifferent epitopes of the same TAA. In some instances, the at least twoantigen binding domains of the first component bind to the same epitopeof the same TAA. In some embodiments, the at least two antigen bindingdomain of the first component bind to different TAAs.

In some embodiments, the second component includes one or more copies ofan antigen-binding domain. In certain embodiments, the second componentcontains at least two antigen binding domains, such as two antigenbinding domains. In some embodiments, the at least two antigen bindingdomains of the second component bind to the same TAA. In some cases, theat least two antigen binding domains of the second component bind todifferent epitopes of the same TAA. In some instances, the at least twoantigen binding domains of the second component binds to a same epitopeof the same TAA. In some embodiments, the at least two antigen bindingdomains of the second component bind to different TAAs.

In some embodiments, the first component contains a first antigenbinding domain and the antigen binding domain of the second component isa second antigen binding domain. In some embodiments, the multispecificantigen binding domain comprises at least a first antigen binding domainand a second antigen binding domain, wherein the first antigen bindingdomain and second antigen binding domain bind to the same TAA. In somecases, the first antigen binding domain and the second antigen bindingdomain bind different epitopes of the same TAA. In some instances, thefirst antigen binding domain and the second antigen binding domain bindthe same epitope of the same TAA. In some embodiments, the multispecificantigen binding domain comprises at least a first antigen binding domainand a second antigen binding domain wherein the first antigen bindingdomain and the second antigen binding domain bind different TAAs.

In some embodiments, the antigen binding domain of the second component(which in some cases is the second antigen binding domain) and the CD3binding region are operably linked via one or more further amino acidlinkers (referred to herein as an intra-component linker). Theintra-component peptide linker of the second component (also called LP2)can be a peptide linker such as any as described in Section 11.3. Theintra-component linker of present in the second component, i.e. linkingthe CD3 binding region and an antigen binding domain, can be of variouslengths, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20 amino acids in length. In some embodiments, the intra-componentlinker of the second component is composed predominately of the aminoacids Glycine and Serine, denoted as GS-linkers herein. In someembodiments, the GS-linker comprises an amino acid sequence selectedfrom the group consisting of GGSGGS, i.e., (GGS)₂ (SEQ ID NO: 10);GGSGGSGGS, i.e., (GGS)₃ (SEQ ID NO: 11); GGSGGSGGSGGS, i.e., (GGS)₄ (SEQID NO: 12); and GGSGGSGGSGGSGGS, i.e., (GGS)₅ (SEQ ID NO: 13).

Provided herein is a multispecific polypeptide construct, themultispecific polypeptide construct comprising a first componentcomprising a heterodimeric Fc region and a second component comprisingan anti-CD3 antibody or antigen-binding fragment comprising a variableheavy chain region (VH) and a variable light chain region (VL), wherein:the VH and VL that comprise the anti-CD3 antibody or antigen bindingfragment are linked to opposite polypeptides of the heterodimeric Fc;the first and second components are coupled by a linker, wherein theheterodimeric Fc region is positioned N-terminal to the anti-CD3antibody or antigen-binding fragment; and one or both of the first andsecond components comprises an antigen binding domain that binds a tumorassociated antigen (TAA).

In some embodiments, the linker is a polypeptide of up to 50 amino acidsin length. In some embodiments, the linker is a polypeptide of up to 25amino acids in length. In some embodiments, the linker is a polypeptideof up to 15 amino acids in length.

In any of the provide embodiments, the one or more antigen bindingdomain that binds TAA results in monovalent, bivalent, trivalent, ortetravalent binding to the TAA. In some embodiments, the one or moreantigen binding domains that bind TAA independently are selected from ansdAb, an scFv or a Fab. In some embodiments, the one or more antigenbinding domains that binds a TAA is a single chain molecule, such as asingle chain antibody fragment containing a VH and a VL, for example ansdAb or an scFv. In some embodiments the one or more antigen bindingdomains that binds a TAA is a sdAb, such as a V_(H)H or a VH_(NAR). Insome embodiments, at least one of the antigen binding domains is a Fabcontaining a first chain comprising a VH-CH1 (Fd) and a second chaincomprising a VL-CL.

In some embodiments, the antigen binding domain that binds the TAA isattached to the VH of the anti-CD3 binding domain. In some embodiments,the antigen binding domain that binds the TAA is attached to the sameside (e.g., knob or hole) of the heterodimeric Fc to which the VH of theanti-CD3 binding domain is attached. In some embodiments, the antigenbinding domain that binds the TAA is a sdAb attached to the VH of theanti-CD3 binding domain. In some embodiments, the antigen binding domainthat binds the TAA is a sdAb attached to same side (e.g., knob or hole)of the heterodimeric Fc domain to which the VH of the anti-CD3 bindingdomain is attached. In some embodiments, the antigen binding domain thatbinds the TAA is a V_(H)H or a VH_(NAR) that is attached to the VH ofthe anti-CD3 binding domain. In some embodiments, the antigen bindingdomain that binds the TAA is a V_(H)H or a VH_(NAR) that is attached tothe same side (e.g., knob or hole) of the heterodimeric Fc domain towhich the VH of the anti-CD3 binding domain is attached. In someembodiments, the antigen binding domain that binds the TAA is a V_(H)Hattached to the VH of the anti-CD3 binding domain. In some embodiments,the antigen binding domain that binds the TAA is a V_(H)H attached tothe same side (e.g., knob or hole) of the heterodimeric Fc domain towhich the VH of the anti-CD3 binding domain is attached. In someembodiments, the antigen binding domain that binds the TAA is a VH_(NAR)attached to the VH of the anti-CD3 binding domain. In some embodiments,the antigen binding domain that binds the TAA is a VH_(NAR) attached tothe same side (e.g., knob or hole) or the Fc domain to which the VH ofthe anti-CD3 binding domain is attached.

In some embodiments, the multispecific polypeptide construct comprisesat least (i) a first polypeptide comprising the first Fc polypeptide ofthe heterodimeric Fc region, the linker and the VH domain of theanti-CD3 antibody or antigen binding fragment; and (ii) a secondpolypeptide comprising the second Fc polypeptide of the heterodimeric Fcregion, the linker and the VL domain of the anti-CD3 antibody or antigenbinding fragment, wherein one or both of the first and secondpolypeptide comprise at least one antigen-binding domain that binds to atumor associated antigen (TAA). In some instances, only one of the firstor second polypeptide comprises the at least one antigen-binding domainthat binds a TAA.

In some embodiments, the at least one of the antigen binding domain(s)is a Fab. In some embodiments, the multispecific polypeptide constructcomprises: (i) a first polypeptide comprising the first Fc polypeptideof the heterodimeric Fc region, the linker and the VH domain of theanti-CD3 antibody or antigen binding fragment; (ii) a second polypeptidecomprising the second Fc polypeptide of the heterodimeric Fc region, thelinker and the VL domain of the anti-CD3 antibody or antigen bindingfragment, and (iii) a third polypeptide comprising a VH-CH1 (Fd) orVL-CL of a Fab antibody fragment that binds to a tumor-associatedantigen, wherein the first and/or second polypeptide further comprisesthe other of the VH-CH1 (Fd) or VL-CL of the Fab antibody fragment. Insome cases, only one of the first or second polypeptide comprises theother of the VH-CH1 (Fd) or VL-CL of the Fab antibody fragment. In someembodiments, both the first or second polypeptide comprises the other ofthe VH-CH1 (Fd) or VL-CL of the Fab antibody fragment. In some cases,the other of the VH-CH1 (Fd) or VL-CL of the Fab antibody fragment ispositioned amino-terminally relative to the Fc region and/orcarboxy-terminally relative to the CD3 binding region of one of thefirst or second polypeptide of the multispecific polypeptide construct.In some embodiments, the other of the VH-CH1 (Fd) or VL-CL of the Fabantibody fragment is positioned amino-terminally relative to the Fcregion of the first polypeptide or second polypeptide andcarboxy-terminally relative to the CD3 binding region of the other ofthe first or second polypeptide.

In some embodiments, the at least one antigen binding domain ispositioned amino-terminally relative to the Fc region and/or ispositioned carboxy-terminally relative to the CD3 binding region of oneof the first or second polypeptide of the multispecific polypeptideconstruct. In some cases, the at least one antigen binding domain ispositioned amino-terminally relative to the Fc region of themultispecific construct and the second antigen binding domain ispositioned carboxy-terminally relative to the CD3 binding region of themultispecific construct. In particular embodiments of providedmultispecific polypeptide constructs, at least one antigen bindingdomain is positioned carboxy-terminally relative to the CD3 bindingregion of the multispecific polypeptide construct. In some embodiments,the at least one antigen binding domain is a sdAb. In some embodimentsthe at least one antigen binding domain that is a sdAb is positionedcarboxy-terminally to the CD3 binding region of the multispecificconstruct. In some embodiments the at least one antigen binding domainthat is a sdAb is positioned amino-terminally to the Fc region of themultispecific construct. In some embodiments the at least one antigenbinding domain is a V_(H)H. In some embodiments, the at least oneantigen binding domain that is a V_(H)H is positioned carboxy-terminallyto the CD3 binding region of the multispecific construct. In someembodiments the at least one antigen binding domain that is a V_(H)H ispositioned amino-terminally to the Fc region of the multispecificconstruct.

In some embodiments, the multispecific polypeptide construct comprises afirst linking peptide (LP1) between the first antigen binding domain andthe immunoglobulin Fc polypeptide region (Fc region). In someembodiments, the multispecific polypeptide construct comprises a secondlinking peptide (LP2) between the anti-CD3 binding domain (CD3 bindingregion) and the second antigen binding domain. In some embodiments, themultispecific polypeptide construct comprises a first linking peptide(LP1) between the first antigen binding domain and the immunoglobulin Fcpolypeptide region (Fc region) and a second linking peptide (LP2)between the anti-CD3 binding domain (CD3 binding region) and the secondantigen binding domain.

In some embodiments, the multispecific polypeptide construct has thestructural arrangement from N-terminus to C-terminus as follows: firstantigen binding domain-LP1-immunoglobulin Fc polypeptide linker region(Fc region)-linker-anti-CD3 binding domain-LP2-second antigen bindingdomain. In some embodiments, the multispecific polypeptide construct hasthe structural arrangement from N-terminus to C-terminus as follows:second antigen binding domain-LP2-immunoglobulin Fc polypeptide linkerregion (Fc region)-linker-anti-CD3 binding domain (CD3 bindingregion)-LP1-first antigen binding domain.

In some embodiments, the two linking peptides LP1 and LP2 are notidentical to each other. In some cases, LP1 or LP2 is independently apeptide of about 1 to 20 amino acids in length. In some examples, LP1 orLP2 independently comprise a peptide that is or comprises any Gly-Serlinker as set forth in SEQ ID NOs: 10-13, 119, 135, 147, 149.

In some embodiments, the multispecific construct is a construct havingany of the structural arrangement shown in FIG. 1. In some embodiments,the construct is a bispecific construct that has a structuralarrangement from N-terminus to C-terminus as follows. The N-terminal endof the bispecific construct includes a first antigen binding domain thatbinds a tumor associated antigen (TAA). The first binding domain binds afirst epitope on the TAA target. Coupled to the first antigen bindingdomain is a central immunoglobulin Fc polypeptide region that regulatesFcγR interactions and/or FcRn interaction. In some embodiments, thecentral immunoglobulin Fc polypeptide region is heterodimeric. Theimmunoglobulin Fc polypeptide region is coupled to a linker located at aposition C-terminal to the end of the immunoglobulin Fc polypeptideregion. The linker is attached to an anti-CD3 binding sequence locatedC-terminal to the Fc region, which, in some cases, is at the distal endof the second component. The C-terminus of the bispecific constructincludes a second antigen binding domain that binds a TAA. In someembodiments, the second antigen binding domain binds the same TAA as thefirst antigen binding domain located within the first component. In someembodiments, the second antigen binding domain binds a second epitope onthe TAA, wherein the second epitope is non-competitive with the firstepitope on the TAA. In some embodiments, the second antigen bindingdomain binds a distinct TAA from that of the first antigen bindingdomain.

In some of any of the provided embodiments, the anti-CD3 antibody orantigen binding fragment is an Fv antibody fragment. In someembodiments, the Fv antibody fragment comprises a disulfide stabilizedanti-CD3 binding Fv fragment (dsFv). In some embodiments, the anti-CD3binding sequence is an Fv antibody fragment that is engineered toinclude a disulfide linkage between the variable heavy chain (VH) andvariable light chain (VL) regions, thereby producing a disulfidestabilized anti-CD3 binding Fv fragment (dsFv). In some embodiments, theVH and VL domains that comprise the anti-CD3 Fv are operably linked toopposite members of a heterodimeric Fc region. In these embodiments, theanti-CD3 Fv binds CD3 in a monovalent fashion. In aspects as provided,the anti-CD3 dsFv does not engage CD3 unless the multispecificpolypeptide construct is bound to a cognate antigen.

In some embodiments, each of the first antigen binding domain and thesecond antigen binding domain of the bispecific construct includes oneor more copies of an antibody or an antigen-binding fragment thereof. Insome embodiments, each of the first antigen binding domain and thesecond antigen binding domain of the bispecific construct includes oneor more copies of an antibody or an antigen-binding fragment thereofselected from the group consisting of a Fab fragment, a F(ab′)₂fragment, an Fv fragment, a scFv, a scAb, a dAb, a single domain heavychain antibody, and a single domain light chain antibody. In someembodiments, the antigen binding domain, or independently each of theantigen binding domains, is an antibody or antigen-binding fragmentthereof selected from the group consisting of a Fab fragment, a F(ab′)2fragment, an Fv fragment, a scFv, a scAb, a dAb, a single domain heavychain antibody, and a single domain light chain antibody. In someembodiments, the each of the first antigen binding domain and the secondantigen binding domain of the bispecific construct includes one or morecopies of one or more single domain antibody (sdAb) fragments, forexample V_(H)H, V_(NAR), engineered V_(H) or V_(K) domains. V_(H)Hs canbe generated from natural camelid heavy chain only antibodies,genetically modified rodents that produce heavy chain only antibodies,or naïve/synthetic camelid or humanized camelid single domain antibodylibraries. V_(NAR)5 can be generated from cartilaginous fish heavy chainonly antibodies. Various methods have been implemented to generatemonomeric sdAbs from conventionally heterodimeric V_(H) and V_(K)domains, including interface engineering and selection of specificgermline families.

In some embodiments, the antibody or antigen-binding fragment is ansdAb. In some cases, the sdAb is a human or humanized sdAb. In someaspects, the sdAb is V_(H)H, V_(NAR), an engineered VH domain or anengineered VK domain. In some examples, the antibody or antigen-bindingfragment thereof is an scFv. In some cases, the antibody orantigen-binding fragment thereof is a Fab.

In some of any of the provided embodiments, the anti-CD3 antibody orantigen-binding fragment comprises a VH CDR1 comprising the amino acidsequence TYAMN (SEQ ID NO: 16); a VH CD2 comprising the amino acidsequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 17); a VH CDR3 comprising theamino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 18), a VL CDR1 comprisingthe amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 19); a VL CDR2comprising the amino acid sequence GTNKRAP (SEQ ID NO: 20); and a VLCDR3 comprising the amino acid sequence ALWYSNLWV (SEQ ID NO: 21).

In some of any of the provided embodiments, the anti-CD3 antibody orantigen-binding fragment comprises a VH CDR1 comprising the amino acidsequence GFTFNTYAMN (SEQ ID NO: 211); a VH CDR2 comprising the aminoacid sequence RIRSKYNNYATY (SEQ ID NO:212); a VH CDR3 comprising theamino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 18), a VL CDR1 comprisingthe amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 19); a VL CDR2comprising the amino acid sequence GTNKRAP (SEQ ID NO: 20); and a VLCDR3 comprising the amino acid sequence ALWYSNLWV (SEQ ID NO: 21).

In some of any of the provided embodiments, the anti-CD3 antibody orantigen-binding fragment comprises a VH CDR1 sequence that includes atleast the amino acid sequence GFTFNTYAMN (SEQ ID NO: 211); a VH CDR2sequence that includes at least the amino acid sequence RIRSKYNNYATY(SEQ ID NO: 212); a VH CDR3 sequence that includes at least the aminoacid sequence HGNFGNSYVSWFAY (SEQ ID NO: 18), a VL CDR1 sequence thatincludes at least the amino acid sequence GSSTGAVTTSNYAN (SEQ ID NO:229); a VL CDR2 sequence that includes at least the amino acid sequenceGTNKRAP (SEQ ID NO: 230); and a VL CDR3 sequence that includes at leastthe amino acid sequence ALWYSNHWV (SEQ ID NO: 225).

In some of any of the provided embodiments, the anti-CD3 antibody orantigen-binding fragment comprises: a VH having the amino acid sequenceof any of SEQ ID NOS: 14, 32-62, 196-198, and 211 or a sequence thatexhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity to any of SEQ ID NOS: 14, 32-62, 196-198, and 211; anda VL having the amino acid sequence of any of SEQ ID NOS: 15, 63-81,191, 199, 200, and 212 or a sequence that exhibits at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQID NOS: 15, 63-81, 191, 199, 200, and 212.

In some embodiments, the anti-CD3 antibody or antigen-binding fragmentis an Fv. In some embodiments, the anti-CD3 Fv comprises: a VH havingthe amino acid sequence of any of SEQ ID NOS: 14, 32-43, 45-47, 48, 196and 211 or a sequence that exhibits at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOS: 14,32-43, 45-47, 48, 196 and 211; and a VL having the amino acid sequenceof any of SEQ ID NOS: 15, 63, 65-71, 73, 75, 77, and 199 or a sequencethat exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity to any of SEQ ID NOS: 15, 63, 65-71, 73, 75, 77,and 199. In some cases, the anti-CD3 dsFv comprises the amino acidsequence of SEQ ID NO: 14 and the amino acid sequence of SEQ ID NO: 15.In other cases, the anti-CD3 Fv comprises the amino acid sequence of SEQID NO: 196 and the amino acid sequence of SEQ ID NO:199.

In some embodiments, the VH and VL chain regions of the CD3 bindingdomain each independently comprise at least one amino acid modification.In some embodiments, the at least one amino acid modification of the VHand VL chain regions of the CD3 binding domain increase the stability ofthe CD3 binding domain. In some embodiments, the at least one amino acidmodification of the VH and VL chain regions of the CD3 binding domainincrease the ability of the CD3 binding domain to bind CD3. In someembodiments, the at least one amino acid modification of the VH and VLchain regions of the CD3 binding domain increase the stability of theCD3 binding domain by creating a disulfide linkage between the VH and VLchain regions.

In some embodiments, the CD3 binding region has a disulfide stabilizedlinkage between the VH and VL regions. In some embodiments, the anti-CD3antibody or antigen-binding fragment is disulfide stabilized Fv (dsFv).In some embodiments, the disulfide stabilized anti-CD3 Fv comprises ananti-CD3 VH with the mutation 44 to Cys and an anti-CD3 VL with themutation 100 to Cys by Kabat numbering. In some embodiments, thedisulfide stabilized anti-CD3 Fv comprises an anti-CD3 VH with themutation G44C and an anti-CD3 VL with the mutation G100C by Kabatnumbering. In some embodiments, the disulfide stabilized anti-CD3 Fvcomprises an anti-CD3 VH with the mutation at position 105 to Cys and ananti-CD3 VL with the mutation position 43 to Cys by Kabat numbering.

In some embodiments, the anti-CD3 dsFv comprises: a VH having the aminoacid sequence of any of SEQ ID NOS: 44, 49-62, 197 and 198 or a sequencethat exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity to any of SEQ ID NOS: 44, 49-62, 197 and 198; anda VL having the amino acid sequence of any of SEQ ID NOS: 64, 72, 74,76, 78-81, 191, 200 and 212 or a sequence that exhibits at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to anyof SEQ ID NOS: 64, 72, 74, 76, 78-81, 191, 200 and 212. In some cases,the anti-CD3 dsFv comprises the amino acid sequence of SEQ ID NO: 44 andthe amino acid sequence of SEQ ID NO: 72. In some embodiments, theanti-CD3 dsFv comprises the amino acid sequence of SEQ ID NO: 198 andthe amino acid sequence of SEQ ID NO: 200. In some embodiments, theanti-CD3 dsFv comprises the amino acid sequence of SEQ ID NO: 197 andthe amino acid sequence of SEQ ID NO: 200.

In some embodiments, the multispecific construct also includes an agentconjugated to the multispecific construct. In some embodiments, theagent is a therapeutic agent. In some embodiments, the agent is adetectable moiety. In some embodiments, the detectable moiety is adiagnostic agent. In some embodiments, the agent is conjugated to themultispecific construct via a linker. In some embodiments, the linker isa non-cleavable linker.

In some embodiments, the multispecific construct described herein isused in conjunction with one or more additional agents or a combinationof additional agents. Suitable additional agents include currentpharmaceutical and/or surgical therapies for an intended application,such as, for example, cancer. For example, the multispecific constructcan be used in conjunction with an additional chemotherapeutic oranti-neoplastic agent.

In some embodiments, the multispecific construct and additional agentare formulated into a single therapeutic composition, and themultispecific construct and additional agent are administeredsimultaneously. In some embodiments, the multispecific construct andadditional agent are separate from each other, e.g., each is formulatedinto a separate therapeutic composition, and the multispecific constructand the additional agent are administered simultaneously, or themultispecific construct and the additional agent are administered atdifferent times during a treatment regimen. For example, themultispecific construct is administered prior to the administration ofthe additional agent, the multispecific construct is administeredsubsequent to the administration of the additional agent, or themultispecific construct and the additional agent are administered in analternating fashion. As described herein, the multispecific constructand additional agent are administered in single doses or in multipledoses.

In some embodiments, the multispecific construct naturally contains oneor more disulfide bonds. In some embodiments, the multispecificconstruct can be engineered to include one or more disulfide bonds.

The disclosure also provides an isolated nucleic acid molecule orpolynucleotide encoding at least a portion of a multispecific constructdescribed herein and/or one or more nucleic acid molecules encoding amultispecific construct described herein, such as for example, at leasta first nucleic acid encoding at least a portion of the first componentof the multispecific construct and a second nucleic acid encoding atleast a portion of the second component of the multispecific construct,as well as vectors that include these isolated nucleic acid sequences.

Among the provided embodiments is a polynucleotide(s) encoding any ofthe provided multispecific polypeptide constructs. Also provided is apolynucleotide encoding a polypeptide chain of any of the providedmultispecific polypeptide constructs. Further provided is apolynucleotide, comprising a first nucleic acid sequence encoding afirst polypeptide of any of the provided multispecific constructs and asecond nucleic acid sequence encoding a second polypeptide of themultispecific construct, wherein the first and second nucleic acidsequence are separated by an internal ribosome entry site (IRES), or anucleic acid encoding a self-cleaving peptide or a peptide that causesribosome skipping. In some cases, the first nucleic acid sequence andsecond nucleic acid sequence are operably linked to the same promoter.In some embodiments, the multispecific polypeptide construct comprises athird polypeptide chain, and the polynucleotide further comprises athird nucleic acid encoding the third polypeptide of the multispecificconstruct. In some embodiments, the third nucleic acid is separated fromthe first and/or second polypeptide by an internal ribosome entry site(IRES), or a nucleic acid encoding a self-cleaving peptide or a peptidethat causes ribosome skipping and/or the third nucleic acid sequence isoperably linked to the same promoter as the first and/or second nucleicacid sequence. In some examples, the nucleic acid encoding aself-cleaving peptide or a peptide that causes ribosome skipping isselected from a T2A, a P2A, a E2A or a F2A (SEQ ID NOS: 159-164, orencoded by the sequence set forth in SEQ ID NO: 165)

Provided herein is a vector comprising any of the providedpolynucleotides. In some embodiments, the vector is an expressionvector. In some examples, the vector is a viral vector or a eukaryoticvector, optionally wherein the eukaryotic vector is a mammalian vector.

Provided is a cell, comprising any of the provided polynucleotides orvectors. In some cases, the cell is recombinant or isolated. In someexamples, the cell is a mammalian cell. In some examples, the cell is aHEK293 or CHO cell.

The disclosure provides methods of producing a multispecific constructby culturing a cell under conditions that lead to expression of themultispecific construct, wherein the cell comprises such a nucleic acidmolecule(s). In some embodiments, the cell comprises such a vector.

Provided herein is a method of producing a multispecific polypeptideconstruct, the method comprising introducing into a cell any of theprovided polynucleotides or vectors and culturing the cell underconditions to that lead to expression of the multispecific construct toproduce the multispecific polypeptide construct. Also provided is amethod of producing a multispecific polypeptide construct, the methodcomprising culturing any of the provided cells under conditions in whichthe multispecific polypeptide is expressed or produced by the cell. Insome cases, the cell is a mammalian cell. In some examples, the cell isa HEK293 or CHO cell. In some embodiments, the method further includesisolating or purifying the multispecific polypeptide construct from thecell. In some cases, the multispecific polypeptide construct is aheterodimer.

Provided herein is a multispecific polypeptide construct produced by anyof the provided methods.

Provided herein is a method of stimulating or inducing an immuneresponse, the method comprising contacting a target cell and a T cellwith the any of the provided multispecific polypeptide constructs orpharmaceutical compositions, said target cell expressing a tumorassociated antigen recognized by the multispecific polypeptideconstruct. In some embodiments, the target cell is a tumor cellexpressing the tumor associated antigen (TAA).

In some embodiments, the contacting is carried out ex vivo or in vitro.In some embodiments, the contacting is carried out in vivo in a subject.

Provided is a method of stimulating or inducing an immune response in asubject, the method comprising administering, to a subject in needthereof, a therapeutically effective amount of any of the providedmultispecific conjugates or pharmaceutical compositions. In some cases,the method increases cell-mediated immunity. In some embodiments, themethod increases T-cell activity. In some embodiments, the methodincreases cytolytic T-cell (CTL) activity. In some examples, the immuneresponse is increased against a tumor or cancer. In some embodiments,the method treats a disease or condition in the subject.

The present disclosure also provides methods of treating, preventing,delaying the progression of or otherwise ameliorating a symptom of oneor more pathologies or alleviating a symptom associated with suchpathologies, by administering a multispecific polypeptide construct ofthe disclosure to a subject in which such treatment or prevention isdesired. Provided herein is a method of treating a disease or conditionin a subject, the method comprising administering, to a subject in needthereof, a therapeutically effective amount of any of the providedmultispecific conjugates or pharmaceutical compositions. In someembodiments, the disease or condition is a tumor or a cancer.

In some embodiments of any of the provided method, the subject, such asthe subject to be treated is, e.g., human or other mammal. In someembodiments of any of the provided method, the subject is a human. Insome embodiments, the subject is a non-human mammal, such as a non-humanprimate, companion animal (e.g., cat, dog, horse), farm animal, workanimal, or zoo animal. In some embodiments, the subject is a rodent.

A multispecific polypeptide construct of the disclosure used in any ofthe embodiments of these methods and uses can be administered at anystage of the disease. For example, such a multispecific polypeptideconstruct can be administered to a patient suffering cancer of anystage, from early to metastatic. The terms subject and patient are usedinterchangeably herein.

A multispecific polypeptide construct of the disclosure used in any ofthe embodiments of these methods and uses can be used in a treatmentregimen comprising neoadjuvant therapy.

A multispecific polypeptide construct of the disclosure used in any ofthe embodiments of these methods and uses can be administered eitheralone or in combination with one or more additional agents, includingsmall molecule inhibitors, other antibody-based therapies, polypeptideor peptide-based therapies, nucleic acid-based therapies and/or otherbiologics. In some embodiments, a multispecific polypeptide construct isadministered in combination with one or more additional agents such as,by way of non-limiting example, a chemotherapeutic agent, such as analkylating agent, an anti-metabolite, an anti-microtubule agent, atopoisomerase inhibitor, a cytotoxic antibiotic, and any other nucleicacid damaging agent. In some embodiments, the additional agent is ataxane, such as paclitaxel (e.g., Abraxane®). In some embodiments, theadditional agent is an anti-metabolite, such as gemcitabine. In someembodiments, the additional agent is an alkylating agent, such asplatinum-based chemotherapy, such as carboplatin or cisplatin. In someembodiments, the additional agent is a targeted agent, such as a kinaseinhibitor, e.g., sorafenib or erlotinib. In some embodiments, theadditional agent is a targeted agent, such as another antibody, e.g., amonoclonal antibody (e.g., bevacizumab), a bispecific antibody, or amultispecific antibody. In some embodiments, the additional agent is aproteasome inhibitor, such as bortezomib or carfilzomib. In someembodiments, the additional agent is an immune modulating agent, such aslenolidominde or IL-2. In some embodiments, the additional agent isradiation. In some embodiments, the additional agent is an agentconsidered standard of care by those skilled in the art. In someembodiments, the additional agent is a chemotherapeutic agent well knownto those skilled in the art. In some embodiments, the multispecificpolypeptide construct and the additional agent(s) are formulated in asingle composition. In some embodiments, the multispecific polypeptideconstruct and the additional agent(s) are administered as two or moreseparate compositions. In some embodiments, the multispecificpolypeptide construct and the additional agent(s) are administeredsimultaneously. In some embodiments, the multispecific polypeptideconstruct and the additional agent(s) are administered sequentially.

In some embodiments, the additional agent(s) is a chemotherapeuticagent, such as a chemotherapeutic agent selected from the groupconsisting of docetaxel, paclitaxel, abraxane (i.e., albumin-conjugatedpaclitaxel), doxorubicin, oxaliplatin, carboplatin, cisplatin,irinotecan, and gemcitabine.

In some embodiments, the additional agent(s) is a checkpoint inhibitor,a kinase inhibitor, an agent targeting inhibitors in the tumormicroenvironment, and/or a T cell or NK agonist. In some embodiments,the additional agent(s) is radiation therapy, alone or in combinationwith another additional agent(s) such as a chemotherapeutic oranti-neoplastic agent. In some embodiments, the additional agent(s) is avaccine, an oncovirus, and/or a DC-activating agent such as, by way ofnon-limiting example, a toll-like receptor (TLR) agonist and/or α-CD40.In some embodiments, the additional agent(s) is a tumor-targetedantibody designed to kill the tumor via ADCC or via direct conjugationto a toxin (e.g., an antibody drug conjugate (ADC).

In some embodiments, the checkpoint inhibitor is an inhibitor of atarget selected from the group consisting of CTLA-4, LAG-3, PD-1, PDL1,TIGIT, TIM-3, B7H3, B7H4, and Vista. In some embodiments, the kinaseinhibitor is selected from the group consisting of B-RAFi, MEKi, and Btkinhibitors, such as ibrutinib. In some embodiments, the kinase inhibitoris crizotinib. In some embodiments, the tumor microenvironment inhibitoris selected from the group consisting of an IDO inhibitor, an α-CSF1Rinhibitor, an α-CCR4 inhibitor, a TGF-beta, a myeloid-derived suppressorcell, or a T-regulatory cell. In some embodiments, the agonist isselected from the group consisting of OX40, GITR, CD137, CD28, ICOS,CD27, and HVEM. In some embodiments, the checkpoint inhibitor is anantibody that binds a target selected from CTLA-4, PD-1, and/or PD-L1.In some embodiments, the checkpoint inhibitor is an anti-CTLA4 antibody,an anti-PD-1 antibody, and an anti-PD-L1 antibody, and/or combinationsthereof. In some embodiments, the checkpoint inhibitor is an anti-CTLA4antibody such as, e.g., Yervoy™ In some embodiments, the checkpointinhibitor is an anti-PD-1 antibody such as, e.g., Opdivo™ and/orKeytruda™.

In some embodiments, the inhibitor is a CTLA-4 inhibitor. In someembodiments, the inhibitor is a LAG-3 inhibitor. In some embodiments,the inhibitor is a PD-1 inhibitor. In some embodiments, the inhibitor isa PDL1 inhibitor. In some embodiments, the inhibitor is a TIGITinhibitor. In some embodiments, the inhibitor is a TIM-3 inhibitor. Insome embodiments, the inhibitor is a B7H3 inhibitor. In someembodiments, the inhibitor is a B7H4 inhibitor. In some embodiments, theinhibitor is a Vista inhibitor. In some embodiments, the inhibitor is aB-RAFi inhibitor. In some embodiments, the inhibitor is a MEKiinhibitor. In some embodiments, the inhibitor is a Btk inhibitor. Insome embodiments, the inhibitor is ibrutinib. In some embodiments, theinhibitor is crizotinib. In some embodiments, the inhibitor is an IDOinhibitor. In some embodiments, the inhibitor is an α-CSF1R inhibitor.In some embodiments, the inhibitor is an α-CCR4 inhibitor. In someembodiments, the inhibitor is a TGF-beta. In some embodiments, theinhibitor is a myeloid-derived suppressor cell. In some embodiments, theinhibitor is a T-regulatory cell.

In some embodiments, the agonist is OX40. In some embodiments, theagonist is GITR. In some embodiments, the agonist is CD137. In someembodiments, the agonist is CD28. In some embodiments, the agonist isICOS. In some embodiments, the agonist is CD27. In some embodiments, theagonist is HVEM.

In some embodiments, the multispecific polypeptide construct isadministered during and/or after treatment in combination with one ormore additional agents such as, for example, a chemotherapeutic agent,an anti-inflammatory agent, and/or a an immunosuppressive agent. In someembodiments, the multispecific polypeptide construct and the additionalagent are formulated into a single therapeutic composition, and themultispecific polypeptide construct and additional agent areadministered simultaneously. Alternatively, the multispecificpolypeptide construct and additional agent are separate from each other,e.g., each is formulated into a separate therapeutic composition, andthe multispecific polypeptide construct and the additional agent areadministered simultaneously, or the multispecific polypeptide constructand the additional agent are administered at different times during atreatment regimen. For example, the multispecific polypeptide constructis administered prior to the administration of the additional agent, themultispecific polypeptide construct is administered subsequent to theadministration of the additional agent, or the multispecific polypeptideconstruct and the additional agent are administered in an alternatingfashion. As described herein, the multispecific polypeptide constructand additional agent are administered in single doses or in multipledoses.

In some embodiments, the multispecific polypeptide construct and theadditional agent(s) are administered simultaneously. For example, themultispecific polypeptide construct and the additional agent(s) can beformulated in a single composition or administered as two or moreseparate compositions. In some embodiments, the multispecificpolypeptide construct and the additional agent(s) are administeredsequentially, or the multispecific polypeptide construct and theadditional agent are administered at different times during a treatmentregimen.

In addition to the elements described above, the multispecificpolypeptide construct can contain additional elements such as, forexample, amino acid sequence N- or C-terminal of the multispecificpolypeptide construct. For example, a multispecific polypeptideconstruct can include a targeting moiety to facilitate delivery to acell or tissue of interest. Multispecific polypeptide construct can beconjugated to an agent, such as a therapeutic agent, a detectable moietyor a diagnostic agent. Examples of agents are disclosed herein.

The multispecific polypeptide construct can also include any of theconjugated agents, linkers and other components described herein inconjunction with a multispecific polypeptide construct of thedisclosure.

The disclosure also pertains to immunoconjugates comprising amultispecific polypeptide construct conjugated to a cytotoxic agent suchas a toxin (e.g., an enzymatically active toxin of bacterial, fungal,plant, or animal origin, or fragments thereof), or a radioactive isotope(i.e., a radioconjugate). Suitable cytotoxic agents for use in targetingdiseased T cells such as in a T cell-derived lymphoma include, forexample, dolastatins and derivatives thereof (e.g. auristatin E, AFP,MMAD, MMAF, MMAE). In some embodiments, the agent is a dolastatin. Insome embodiments, the agent is an auristatin or derivative thereof. Insome embodiments, the agent is a maytansinoid or maytansinoidderivative. In some embodiments, the agent is DM1 or DM4. In someembodiments, the agent is a duocarmycin or derivative thereof. In someembodiments, the agent is a calicheamicin or derivative thereof. In someembodiments, the agent is a pyrrolobenzodiazepine.

In some embodiments, the linker between the multispecific polypeptideconstruct and the cytotoxic agent is cleavable. In some embodiments, thelinker is non-cleavable. In some embodiments, two or more linkers arepresent. The two or more linkers are all the same, e.g., cleavable ornon-cleavable, or the two or more linkers are different, e.g., at leastone cleavable and at least one non-cleavable.

The multispecific polypeptide constructs and conjugates thereof areuseful in methods for treating a variety of disorders and/or diseases.Non-limiting examples of disease include: all types of cancers (breast,lung, colorectal, prostate, melanomas, head and neck, pancreatic, etc.),rheumatoid arthritis, Crohn's disuse, SLE, cardiovascular damage,ischemia, etc. For example, indications would include leukemias,including T-cell acute lymphoblastic leukemia (T-ALL), lymphoblasticdiseases including multiple myeloma, and solid tumors, including lung,colorectal, prostate, pancreatic, and breast, including triple negativebreast cancer. For example, indications include bone disease ormetastasis in cancer, regardless of primary tumor origin; breast cancer,including by way of non-limiting example, ER/PR+ breast cancer, Her2+breast cancer, triple-negative breast cancer; colorectal cancer;endometrial cancer; gastric cancer; glioblastoma; head and neck cancer,such as esophageal cancer; lung cancer, such as by way of non-limitingexample, non-small cell lung cancer; multiple myeloma ovarian cancer;pancreatic cancer; prostate cancer; sarcoma, such as osteosarcoma; renalcancer, such as by way of nonlimiting example, renal cell carcinoma;and/or skin cancer, such as by way of nonlimiting example, squamous cellcancer, basal cell carcinoma, or melanoma. In some embodiments, thecancer is a squamous cell cancer. In some embodiments, the cancer is askin squamous cell carcinoma. In some embodiments, the cancer is anesophageal squamous cell carcinoma. In some embodiments, the cancer is ahead and neck squamous cell carcinoma. In some embodiments, the canceris a lung squamous cell carcinoma.

Also provided is a pharmaceutical composition comprising any of themultispecific polypeptide constructs provided herein and apharmaceutically acceptable carrier. In some cases, the pharmaceuticalcomposition is sterile. Pharmaceutical compositions according to thedisclosure can include a multispecific polypeptide construct of thedisclosure and a carrier. These pharmaceutical compositions can beincluded in kits, such as, for example, diagnostic kits.

One skilled in the art will appreciate that the antibodies of thedisclosure have a variety of uses. For example, the proteins of thedisclosure are used as therapeutic agents for a variety of disorders.The antibodies of the disclosure are also used as reagents in diagnostickits or as diagnostic tools, or these antibodies can be used incompetition assays to generate therapeutic reagents.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic of the basic components of the multispecificpolypeptide constructs of the present disclosure having constrained CD3binding. The antigen binding domain(s) are positioned at the aminoand/or carboxy termini. The Fc region, such as a heterodimeric Fcregion, is positioned N-terminal to the CD3 binding region. Thispositioning of the Fc in close proximity to the CD3 binding regionobstructs CD3 binding. The linker can be a non-cleavable linker asprovided herein.

FIGS. 2A and B is a schematic of various FRa-targeting constrained CD3constructs composed of two polypeptides, Chain 1 and Chain 2. As shownin FIG. 2A, Chain 1 contains a FRa sdAb (antigen binding domain), linkedto a heterodimeric Fc “hole,” linked via a non-cleavable linker (rangingfrom 3 amino acids in cx1356 to 18 amino acids in cx681) to anti-CD3 VLdomain, linked to a second FRα sdAb; and Chain 2 contains a FRα sdAb,linked to a complementary heterodimeric Fc “knob”, linked via the samenon-cleavable linker as above to anti-CD3 VH domain, linked to secondFRα sdAb. When co-expressed the CD3 binding domain is properly assembledvia the association of the VL:VH on the hole and knob, respectively(FIG. 2B). An exemplary anti-CD3 is a disulfide-stabilized Fv (dsFv)containing a variable light (VL) chain with the mutation G100C and avariable heavy (VH) chain with a mutation G44C.

FIG. 3A is a schematic of various B7H3-targeting constrained CD3constructs composed of two polypeptides, Chain 1 and Chain 2. Chain 1contains either a heterodimeric Fc “hole,” linked via a non-cleavablelinker to an anti-CD3 VL domain modified at G100C (top); aB7H3-targeting sdAb linked to a heterodimeric Fc “hole,” linked via anon-cleavable linker to an anti-CD3 VL domain (middle); or anB7H3-targeting sdAb linked to a heterodimeric Fc “hole,” linked via anon-cleavable linker to an anti-CD3 VL domain modified at G100C(bottom). Chain 2 contains either a B7H3-targeted sdAb, linked to acomplementary heterodimeric Fc “knob,” linked via the linker as above toan anti-CD3 VH domain modified at G44C linked to second B7H3 sdAb (top);a B7H3-targeted sdAb, linked to a complementary heterodimeric Fc “knob,”linked via the linker as above to an anti-CD3 VH domain (middle); or aB7H3-targeted sdAb, linked to a complementary heterodimeric Fc “knob,”linked via the linker as above to an anti-CD3 VH domain modified by G44C(bottom). When co-expressed the CD3 binding domain is properly assembledvia the association of the VL:VH on the hole and knob, respectively.Where denoted the VH:VL interaction is stabilized by an engineereddisulfide bond between the modified residues G44C in the VH domain andG100C in the VL domain.

FIG. 3B is a schematic of various B7H3-targeting constrained CD3constructs composed of two polypeptides, Chain 1 and Chain 2. Chain 1contains a heterodimeric Fc “hole,” linked via a non-cleavable linker toan anti-CD3 VL domain modified at G100C linked to a co-stimulatoryreceptor targeting sdAb. Chain 2 contains either a B7H3-targeted sdAb,linked to a complementary heterodimeric Fc “knob,” linked via the linkeras above to an anti-CD3 VH domain modified at G44C linked to secondB7H3-targeted sdAb (top); a heterodimeric Fc “knob,” linked via thelinker as above to an anti-CD3 VH domain modified at G44C linked to aB7H3-targeted sdAb (middle); or a B7H3-targeted sdAb, linked to acomplementary heterodimeric Fc “knob,” linked via the linker as above toan anti-CD3 VH domain modified by G44C (bottom). When co-expressed theCD3 binding domain is properly assembled via the association of theVL:VH on the hole and knob, respectively. VH:VL interaction isstabilized by an engineered disulfide bond between the modified residuesG44C in the VH domain and G100C in the VL domain. The resultingconstructs are engage B7H3 either in bivalent (top) or monovalent(middle and bottom) manner. All the constructs herein express contain aco-stimulatory receptor targeting sdAb.

FIG. 3C is a schematic of various B7H3-targeting constrained CD3constructs composed of three polypeptides, Chain 1, Chain 2 and Chain 3,wherein the B7H3 targeting domain is a FAB. Chain 1 contains aBH73-targeting VH, an IgG Constant Heavy 1 (CH1) linked via a hinge to afirst member of a heterodimeric Fc (Fc-Het-1), linked via the linker asabove to an anti-CD3 VL domain that either lacks (top) or contains themodification of G100C (bottom). Chain 2 contains a BH73-targeting VH, anIgG Constant Heavy 1 (CH1) linked via a hinge to a second member of aheterodimeric Fc (Fc-Het-2), linked via the linker as above to ananti-CD3 VH domain that either lacks (top) or contains the modificationof G44C (bottom). Chain 3 contains a complementary B7H3-targeting VLdomain linked to human Ig Constant Light (CL) region. When co-expressedthe CD3 binding domain is properly assembled via the association of theVL:VH on the complimentary heterodimeric Fc regions. Where denoted theVH:VL interaction is stabilized by an engineered disulfide bond betweenthe modified residues G44C in the VH domain and G100C in the VL domain.

FIG. 4A is a schematic of various DLL3-targeting constrained CD3constructs composed of two polypeptides, Chain 1 and Chain 2. Chain 1contains a heterodimeric Fc “hole”, linked via a non-cleavable linker toan anti-CD3 VL domain modified at G100C linked to a co-stimulatoryreceptor targeting sdAb. Chain 2 contains either a DLL3-targeted sdAb,linked to a complementary heterodimeric Fc “knob”, linked via the linkeras above to an anti-CD3 VH domain modified at G44C linked to secondDLL3-targeted sdAb (top); a heterodimeric Fc “knob”, linked via thelinker as above to an anti-CD3 VH domain modified at G44C linked to aDLL3-targeted sdAb (middle); or a DLL3-targeted sdAb, linked to acomplementary heterodimeric Fc “knob”, linked via the linker as above toan anti-CD3 VH domain modified by G44C (bottom). When co-expressed theCD3 binding domain is properly assembled via the association of theVL:VH on the hole and knob, respectively. VH:VL interaction isstabilized by an engineered disulfide bond between the modified residuesG44C in the VH domain and G100C in the VL domain. The resultingconstructs are engage DLL3 either in bivalent (top) or monovalent(middle and bottom) manner. All the constructs herein express contain aco-stimulatory receptor targeting sdAb.

FIG. 4B is a schematic of a DLL3-targeting constrained CD3 construct,cx5499 composed of two polypeptides, Chain 1 and Chain 2. cx5499 isidentical to cx5352 shown in FIG. 4A (top), but lacking co-stimulatoryreceptor targeting sdAb on the C-terminus of Chain 1. When co-expressedthe CD3 binding domain is properly assembled via the association of theVL:VH on the hole and knob, respectively. VH:VL interaction isstabilized by an engineered disulfide bond between the modified residuesG44C in the VH domain and G100C in the VL domain. The resultingconstructs are engage DLL3 either in bivalent (top) or monovalent(middle and bottom) manner.

FIG. 5A-D depict cellular binding by representative FRa-targetingconstrained CD3 engaging constructs, cx1356 and cx681. FIG. 5A and FIG.5C show binding to Ovcar5 cells (a FRα positive ovarian cancer cellline). FIG. 5B and FIG. 5D depict the lack of binding to T-cells. FIG.5A and FIG. 5B display histograms of the normalized cell counts vsfluorescence at 100 nM of each construct. The secondary anti-human APCantibody only control is shown in the filled black trace, while thepositive control anti-CD3 binding is shown in the open trace, and cx1356and cx681 are shown in the gray shaded traces in FIG. 5A and FIG. 5B.The full titration of each construct on the various cell types are shownin FIG. 5C and FIG. 5D.

FIG. 6A-F depict cellular binding by representative B7H3-targetingconstrained CD3 engaging constructs. FIGS. 6A, C, and E show binding toA375 cells (a B7H3 positive human melanoma cell line). FIGS. 6B, D, andF show the lack of binding to isolated T cells.

FIG. 7A-F depict the impact of linker length on the capacity to agonizeCD3 in the presence of FRα positive IGROV1 cells (FIG. 7A, 7C, 7E), orFRα negative NCI-H460 cells (FIG. 7B, 7D, 7F). FIG. 7A-B show thekinetics of CD3 signaling by 2 nM of various constructs on antigenpositive and negative cells, respectively. FIG. 7C-D show the magnitudeof CD3 agonizing capacity by 2 nM of various constructs on antigenpositive and negative cells, respectively. FIG. 7E-F show the potency ofCD3 agonizing capacity of various constructs with differing linkerlengths on antigen positive and negative cells, respectively. A JurkatCD3 NFAT-GFP reporter cell line was used to assess CD3 signaling.Constrained CD3 binding proteins only effectively engage and cluster CD3on T cells when bound to a second antigen on target cells.

FIG. 8A-D depict the ability of representative B7H3-targetingconstrained CD3 engaging constructs to agonize CD3 in a target dependentmanner. FIG. 8A and FIG. 8C depict the capacity to mediate CD3 signalingin the presence of B7H3 positive A375 cells, while FIG. 8B and FIG. 8Dshow the inability to mediate CD3 signaling in the presence of B7H3negative CCRF-CEM cells. A Jurkat CD3 NFAT-GFP reporter cell line wasused to assess CD3 agonism.

FIG. 9A depicts the ability of a representative B7H3-targetingconstrained CD3 engaging construct (cx3072) to induce T-cell mediatedcytotoxicity in a target dependent manner. Target cells were labeledwith cytoID red label and dying cells were visualized by addition ofCaspase 3/7 green reagent. Cytotoxicity was assessed by determining theoverlap area of red target cells and green dying cells. A B7H3 negativeA375 cell line, generated by CRISPR technology was used to test antigenspecific T-cell mediated cytotoxicity. cx3072 was unable to elicitT-cell mediated cytotoxicity of these B7H3 deficient cells.

FIG. 9B shows the ability of cx5952 to induce T-cell mediatedcytotoxicity in the presence of B7H3 positive A375 cells, but not in thepresence of B7H3 negative CCRF-CEM cells.

FIGS. 10A and 10B depict the ability of representative B7H3-targetingconstrained CD3 engaging constructs to induce T-cell mediatedcytotoxicity in a target dependent manner. FIG. 10A depicts the capacityof these constructs to induce T-cell mediated cytotoxicity in thepresence of B7H3 positive A375 cells, while FIG. 10B depicts thecapacity of these constructs to induce T-cell mediated cytotoxicity inthe presence of B7H3 negative CCRF-CEM cells. Cytotoxicity was assessedby determining the overlap area of red target cells and green dyingcells.

FIGS. 10C and 10D depict the ability of representative B7H3-targetingconstrained CD3 engaging constructs to induce T-cell mediatedcytotoxicity in a target dependent manner. FIG. 10C depicts the capacityof these constructs to induce T-cell mediated cytotoxicity in thepresence of B7H3 positive A375 cells, while FIG. 10C depicts thecapacity of these constructs to induce T-cell mediated cytotoxicity inthe presence of B7H3 negative CCRF-CEM cells. Cytotoxicity was assessedby determining the overlap area of red target cells and green dyingcells.

FIGS. 11A-C depict the ability of a representative B7H3-targetingconstrained CD3 engaging construct (cx5952) to induce T-cell mediatedT-cell activation in a target dependent manner. T-cell activation ofCD4+ or CD8+ T cells was assessed by expression of the T cell activationmarkers CD25 (FIG. 11A), CD69 (FIG. 11B), and CD71 (FIG. 11C).

FIGS. 11D-11K depict the ability of representative B7H3-targetingconstrained CD3 engaging constructs to induce T-cell activation in atarget dependent manner. B7H3-target-dependent CD4+ T-cell activation isshown by expression of the T cell activation markers CD25 (FIG. 11D) andCD71 (FIG. 11F). B7H3-target-dependent CD8+ T-cell activation is shownby expression of the T cell activation markers CD25 (FIG. 11H) and CD71(FIG. 11J). T-cell activation was not observed in the absence of B7H3positive cells, based on T cell activation marker CD25 as shown in CD4+T cells (FIG. 11E) or CD8+ T cells (FIG. 11I) or based on T cellactivation marker CD71 as shown in CD4+ T cells (FIG. 11G) or CD8+ Tcells (FIG. 11K)

FIG. 12A depicts the ability of representative B7H3-targetingconstrained CD3 engaging constructs to induce IFNγ production in atarget dependent manner. FIG. 12A shows the production of IFNγ fromT-cells cultured with B7H3 positive A375 cells and in the presence ofB7H3 negative CCRF-CEM cells in the presence of the representativeB7H3-targeting CD3 engaging constructs.

FIG. 12B depicts the ability of representative B7H3-targetingconstrained CD3 engaging constructs to induce IFNγ production in atarget dependent manner. FIG. 12B shows the production of IFNγ fromT-cells cultured with B7H3 positive A375 cells and in the presence ofB7H3 negative CCRF-CEM cells in the presence of the representativeB7H3-targeting CD3 engaging constructs.

FIGS. 13A and 13B depict cellular binding of representativeB7H3-targeting constrained CD3 engaging constructs. cx5187 and cx5823each contain two B7H3 binding domains while complex cx5873 and cx5965each contain one B7H3 binding domain. FIG. 13A shows binding to B7H3positive A375 cells. FIG. 13B shows the lack of binding to B7H3 negativeCCRF-CEM cells and isolated T-cells.

FIG. 13C and FIG. 13D depict the ability of representativeB7H3-targeting constrained CD3 engaging constructs to agonize CD3 in atarget dependent manner. FIG. 13C shows that engaging B7H3 positive A375cells with a construct hat is bivalent and bi-epitopic to B7H3 (cx5187)induced more potent CD3 signaling than constructs that are monovalent toB7H3 (cx5873 and cx5965). FIG. 13D shows the lack of activation ofT-cells in the presence of B7H3 negative CCRF-CEM cells. A Jurkat CD3NFAT-GFP reporter cell line was used to assess CD3 agonism.

FIG. 14A and FIG. 14B depict the ability of representativeB7H3-targeting constrained CD3 engaging constructs to induce T-cellmediated cytotoxicity in a target dependent manner. FIG. 14A shows thattargeting B7H3 positive A375 cells with a construct that is bivalent andbi-epitopic to B7H3 (cx5187) induced more potent T-cell mediatedcytotoxicity than constructs that are monovalent to B7H3 (cx5873 andcx5965). FIG. 14B depicts the lack of T-cell mediated cytotoxicityagainst B7H3 negative CCRF-CEM cells.

FIG. 15A-D depict the ability of representative B7H3-targetingconstrained CD3 engaging molecules to activate T-cells in the presenceof B7H3 positive A375cells, but not in the presence of B7H3 negativeCCRF-CEM cells. FIGS. 15A and 15B show that targeting B7H3 positive A375cells with a construct that is bivalent and bi-epitopic to B7H3(cx5187), induced more potent CD25 expression on CD4+ and CD8+ T-cellsthan constructs that are monovalent to B7H3 (cx5873 and cx5965). FIGS.15C and 15D show the lack of CD25 expression on CD4+ and CD8+ T-cells inthe presence of B7H3 negative CCRF-CEM cells.

FIGS. 16A and 16B demonstrate the ability of representativeB7H3-targeting constrained CD3 engaging constructs to elicit T-cellmediated cytotoxicity in the presence of B7H3-positive A375 cells (FIG.16A) but not in the presence of CCRF-CEM B7H3-negative cells (FIG. 16B).

FIGS. 16C-16J demonstrate the ability of representative B7H3-targetingconstrained CD3 engaging constructs to elicit T cell activation in thepresence of B7H3-positive A375 cells but not in the presence of CCRF-CEMB7H3-negative cells, as assessed by: expression of CD25 on CD4+ T cells(FIGS. 16C and 16D, respectively), CD25 expression on CD8+ T cells(FIGS. 16E and 16F, respectively), CD71 expression on CD4+ T cells(FIGS. 16G and 1611, respectively), CD71 expression on CD8+ T cells(FIGS. 161 and 16J, respectively).

FIGS. 16K and 16L demonstrate the ability of representativeB7H3-targeting constrained CD3 engaging constructs to elicit T cellcytokine production in the presence of B7H3-positive A375 cells (FIG.16K) but not in the presence of CCRF-CEM B7H3-negative cells (FIG. 16L).

FIGS. 17A and 17B demonstrate that representative monovalent (cx5800 andcx5801) and bivalent (cx5352) DLL3-targeting constrained CD3 engagingconstructs bound to a DLL3 expressing cell line, SHP-77 (FIG. 17A), butnot to isolated T-cells (FIG. 17B). Binding was assessed by flowcytometry.

FIG. 17C depicts the ability of representative DLL3-targetingconstrained CD3 engaging constructs to agonize CD3 signaling in thepresence of DLL3 positive SHP-77 cells. Engaging DLL3 positive cellswith a construct that is bivalent and bi-epitopic to DLL3 (cx5352)induced more potent T-cell activation than constructs that aremonovalent to DLL3 (cx5800 and cx5801). A Jurkat CD3 NFAT-Luciferasereporter cell line was used to assess CD3 signaling.

FIG. 18A-18E demonstrate the ability of a representative DLL3-targetingconstrained CD3 engaging construct, cx5499 to elicit T-cell mediatedcytotoxicity and T-cell activation in the presence of DLL3-positiveSHP-77 cells. FIG. 18A demonstrates the ability of the representativeDLL3-targeting constrained CD3 engaging constructs to elicit T-cellmediated cytotoxicity in the presence of DLL3-positive SHP-77 cells.FIGS. 18C-18D demonstrate the ability of representative DLL3-targetingconstrained CD3 engaging constructs to elicit T cell activation in thepresence of DLL3-positive SHP-77 cells, as assessed by: expression ofCD25 on CD4+ T cells (FIG. 18B), CD69 expression on CD4+ T cells (FIG.18C), CD25 expression on CD8+ T cells (FIG. 18D) and CD69 expression onCD8+ T cells (FIG. 18E).

DETAILED DESCRIPTION

The present disclosure provides constrained T-cell engaging fusionproteins in the form of multispecific polypeptide constructs that bindat least CD3 and a second antigen. The multispecific polypeptideconstructs provided herein include at least a first component thatincludes one or more copies of an antigen-binding domain that binds anantigen operably linked to an immunoglobulin Fc region, a secondcomponent that includes one or more copies of at least a binding domainthat binds CD3 (referred to herein as an anti-CD3 binding domain or aCD3 binding region, which are terms that are used interchangeablyherein), and a linker, such as a polypeptide linker, that joins thefirst component and the second component. In some embodiments, theantigen is a tumor associated antigen (TAA). In some embodiments, thelinker is a non-cleavable linker. In some embodiments, the linker doesnot contain a substrate recognition site that is specifically recognizedby a protease, such as a protease that is granzyme B, an MMP ormatriptase.

The provided multispecific polypeptide constructs include aconfiguration in which the first component containing the Fc region isN-terminal to the second component containing the CD3 binding region. Insuch an embodiment, the first and second components are joined via alinker that is C-terminal to the end of the Fc region. In someembodiments the antigen binding domain(s) is positioned on theamino-terminal (N-term) region of the multispecific polypeptideconstruct. In some embodiments, the antigen binding domain(s) ispositioned on the carboxy-terminal (C-term) region of the multispecificpolypeptide construct. In some embodiments, the antigen bindingdomain(s) is positioned on both the N- and C-terminal regions of themultispecific polypeptide construct. Various configurations of amultispecific polypeptide construct as provided herein are shown in FIG.1.

The provided multispecific polypeptide constructs exhibit constrainedT-cell engaging activity because such constructs only substantially bindto CD3 once an antigen is bound via the antigen-binding domain. This isexemplified in the Examples and Figures provided herein, whichdemonstrate the ability of constrained CD3-engaging proteins toefficiently bind TAA positive cells, while having little to no bindingof T cells. This unique property allows constrained CD3-engagingproteins to distribute to sites where TAA is present without binding toperipheral T cells. This format is distinct from other CD3 engagingmultispecific constructs, in that constitutive CD3 binding is disallowedor eliminated, providing a significant benefit by avoiding peripheralT-cell binding and permitting superior distribution to the site(s) whereantigen is present as recognized by the antigen binding domain.Furthermore, other CD3 engaging constructs mediate antigen-dependentT-cell activation. However, the multispecific polypeptide constructsprovided herein mediate both antigen dependent T-cell binding andactivation.

The constrained T-cell engaging activity of the provided multispecificpolypeptide constructs is due, in some aspects, to the positioning ofthe Fc region N-terminal to the CD3-binding region. In some embodiments,such positioning reduces, attenuates, dampens and/or prevents CD3binding by the CD3 binding region. In the absence of antigen binding bythe antigen binding domain, the multispecific polypeptide constructsprovided herein demonstrate reduced or eliminated CD3 binding and T-cellactivating capacity. In some embodiments, in the presence of an antigenbinding event mediated by the antigen binding domain(s) of themultispecific polypeptide constructs, the capacity to bind CD3 by theCD3 binding region is greatly enhanced. In some embodiments, in thepresence of an antigen binding event mediated by the antigen bindingdomains(s) of the multispecific polypeptide constructs, the capacity toactivate T-cells is greatly enhanced. Engagement of its cognate antigenby the antigen binding domain(s) within the multispecific polypeptideconstruct leads to subsequent T-cell engagement and mediatesantigen-dependent T-cell activation, such as cytotoxicity, cytokinerelease, degranulation and proliferation. In some embodiments, theprovided multispecific polypeptide constructs can be used to increase animmune response, such as to enhance T-cell activity, including cytolytic(or cytotoxic) T-cell activity. The modulation of the immune responsecan, in some aspects, treat a disease or condition in a subject.

In some embodiments, the one or more antigen binding domains bind anantigen on a tumor cell or a cell of the tumor microenvironment. In someaspects, the provided multispecific polypeptide constructs can be usedto increase immune responses, such as T-cell activity, e.g. cytotoxicityactivity, against a tumor or cancer. In some embodiments, the providedmultispecific polypeptide constructs can be used to treat a tumor orcancer in the subject.

In some embodiments, the multispecific polypeptide constructs of thedisclosure ensure that there will be no binding of T-cells via CD3 inperipheral blood, as the CD3 binding region of these constructs isconstrained or otherwise blocked and/or inhibited by the presence of theFc region. Thus, the multispecific polypeptide constructs of thedisclosure provide a number of advantages. In some aspects, theseconstructs limit the sink effect caused by binding all T-cells. In someaspects, these constructs reduce systemic toxicity.

In some embodiments, the provided multispecific polypeptide constructsof the disclosure allow for controlled biodistribution to a desired sitein a subject, such as, for example, a site of tumor-associated antigen(TAA) expression. Sites of TAA expression include, for example, tumorand the surrounding tumor microenvironment.

In some embodiments, the multispecific polypeptide constructs of thedisclosure exhibit specificity for CD3 and one or more other antigen. Insome embodiments, the multispecific polypeptide constructs can containmore than one antigen binding domain able to bind one or more TAA, suchas 2, 3 or 4 antigen binding domains, see e.g. FIG. 1. In someembodiments, the one or more antigen binding domains bind the sameantigen. In some embodiments, the multispecific polypeptide constructsinclude more than one antigen binding domains that bind distinctepitopes on the same antigen. In some embodiments, the multispecificpolypeptide constructs include more than one antigen binding domainsthat bind one or more distinct antigens. In some embodiments, themultispecific polypeptide constructs include more than one antigenbinding domains that bind distinct epitopes on the same antigens as wellas include additional antigen binding domains that bind to one or moredistinct antigens. In some aspects, the provided multispecificpolypeptide constructs are bispecific polypeptide constructs, such thatthey are able to bind to CD3 and to another antigen, such as a TAA, viabinding of the antigen-binding domain of the multispecific polypeptideconstruct. In some examples, the provided multispecific polypeptideconstructs are bispecific polypeptide constructs that providemultivalent engagement of one or more TAA through the use of a firstantigen-binding domain and a second antigen-binding domain. For example,in some embodiments, the bispecific polypeptide constructions include afirst antigen-binding single domain antibody (sdAb) and a secondantigen-binding sdAb.

In some embodiments, the multispecific polypeptide constructs providedherein exist in two states in terms of capacity to bind CD3 andsubsequently activate T-cells: (1) the “inactive” state occurs whenthere is no binding of any or all of the antigen binding domain(s), suchthat the CD3 binding is constrained and T-cell interaction is obviated,and (2) the “active” state occurs upon antigen binding by any or all ofthe antigen binding domain(s), such that the CD3 binding region is ableto bind CD3 and the T-cell interaction is allowed.

In some embodiments, the Fc region is linked to the CD3 binding domainvia a linker or linkers. In some embodiments, the Fc region is linked tothe CD3 binding region via a non-cleavable linker or linkers, such asany as described.

In some embodiments, the Fc region is a homodimeric Fc region. In someembodiments, the Fc region is a heterodimeric Fc region. In someembodiments, the Fc region is a monomeric Fc region. In someembodiments, the Fc region of the multispecific polypeptide constructsare capable of interacting with FcγRs and mediating innate immuneeffector functions, for example, antibody dependent cellular toxicity(ADCC) and antibody dependent cellular phagocytosis (ADCP). In someembodiments, the Fc region of the multispecific polypeptide constructsare capable of interacting with complement proteins, namely C1q, andmediating complement dependent cytotoxicity. Thus, in some aspects, themultispecific polypeptide constructs of the disclosure allow formultiple immune effector mechanisms, including innate immune effectorsand T-cells.

In some embodiments, the multispecific polypeptide constructs of thedisclosure allow for T-cell and NK cell mediated cytotoxicity to occursimultaneously. In some cases, such activity can occur in amultispecific polypeptide construct in which is contained a firstantigen binding domain, e.g., a first anti-TAA antigen binding domain,and a second antigen binding domain, e.g., a second anti-TAA antigenbinding domain, that can target distinct and/or non-competing epitopeson a given TAA.

It is contemplated that the constrained CD3 engaging constructs areamenable for use with any TAA-binding domain, allowing bettertherapeutic exposure within the tumor or tumor-microenvironment byavoiding interactions with peripheral T-cells and mediating potentTAA-dependent T-cell cytotoxicity. In some embodiments, the secondportion or component contains a CD3 binding region that is monovalent toCD3, such that there will be no activation of T-cell unless there is TAApresent.

In some aspects, the multispecific polypeptide constructs of thedisclosure provide a number of advantages over current bispecifictherapeutics. The multispecific polypeptide constructs of the disclosureare smaller than a conventional therapeutic antibody, e.g., 150 kDa vs.125 kDa, which will allow for better target, e.g. tumor, penetration. Insome aspects, the size of the entire multispecific polypeptide constructprovides long half-life for the construct. In some aspects, themultispecific polypeptide constructs of the disclosure exhibit reducedsystemic toxicity or toxicity of any area outside the tumor and/or tumormicroenvironment, since CD3 binding by the CD3 binding region depends onTAA engagement before CD3 engagement will occur.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

I. DEFINITIONS

Unless otherwise defined, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. The term “a”entity or “an” entity refers to one or more of that entity. For example,a compound refers to one or more compounds. As such, the terms “a”,“an”, “one or more” and “at least one” can be used interchangeably.Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular.Generally, nomenclatures utilized in connection with, and techniques of,cell and tissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

As used herein, the term “antibody” refers to immunoglobulin moleculesand antigen-binding portions of immunoglobulin (Ig) molecules, i.e.,molecules that contain an antigen binding site that specifically binds(immunoreacts with) an antigen. By “specifically bind” or “immunoreactswith” or “immunospecifically bind” is meant that the antibody reactswith one or more antigenic determinants of the desired antigen and doesnot react with other polypeptides or binds at much lower affinity(K_(d)>10⁻⁶). Antibodies include, but are not limited to, polyclonal,monoclonal, chimeric, fully human, domain antibody, single chain, Fab,and F(ab′)₂ fragments, Fvs, scFvs, and a Fab expression library.Typically, an “antigen-binding fragment” contains at least one CDR of animmunoglobulin heavy and/or light chain that binds to at least oneepitope of the antigen of interest. In this regard, an antigen-bindingfragment may comprise 1, 2, 3, 4, 5, or all 6 CDRs of a variable heavychain (VH) and variable light chain (VL) sequence from antibodies thatbind the antigen, such as generally six CDRs for an antibody containinga VH and a VL (“CDR1,” “CDR2” and “CDR3” for each of a heavy and lightchain), or three CDRs for an antibody containing a single variabledomain. Antigen binding fragments include single domain antibodies, suchas those only containing a VH or only containing a VL, including, forexample, V_(H)H, V_(NAR), engineered V_(H) or V_(K) domains.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Ingeneral, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, IgG₃, IgG₄, and others.Furthermore, in humans, the light chain may be a kappa chain or a lambdachain.

The term “monoclonal antibody” (mAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

The term “antigen-binding site” or “binding portion” refers to the partof the immunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains, referred to as “hypervariable regions,” are interposed betweenmore conserved flanking stretches known as “framework regions,” or“FRs”. Thus, the term “FR” refers to amino acid sequences that arenaturally found between, and adjacent to, hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.” Theassignment of amino acids to each domain is in accordance with thedefinitions of Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987 and 1991)), orChothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature342:878-883 (1989).

As used herein, the term “epitope” includes any specific portion of anantigen targeted by an antibody, antibody fragment or other bindingdomain. The term “epitope” includes any protein region to which specificbinding is directed. The term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin or T-cell receptor.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics. For example, antibodies may beraised against N-terminal, central, or C-terminal peptides of apolypeptide. In addition, antibodies may be raised against linear ordiscontinuous epitopes of a polypeptide. An antibody is said tospecifically bind an antigen when the dissociation constant is ≤1 μM;for example, in some embodiments ≤100 nM and in some embodiments, ≤10 nMand does not display binding to other proteins either closely related ordistinct.

As used herein, the terms “specific binding,” “immunological binding,”and “immunological binding properties” refer to the non-covalentinteractions of the type that occur between an immunoglobulin moleculeand an antigen for which the immunoglobulin is specific. The strength,or affinity of immunological binding interactions can be expressed interms of the dissociation constant (K_(d)) of the interaction, wherein asmaller K_(d) represents a greater affinity. Immunological bindingproperties of selected polypeptides can be quantified using methods wellknown in the art. One such method entails measuring the rates ofantigen-binding site/antigen complex formation and dissociation, whereinthose rates depend on the concentrations of the complex partners, theaffinity of the interaction, and geometric parameters that equallyinfluence the rate in both directions. Thus, both the “on rate constant”(K_(m)) and the “off rate constant” (K_(off)) can be determined bycalculation of the concentrations and the actual rates of associationand dissociation. (See Nature 361:186-87 (1993)). The ratio ofK_(off)/K_(on) enables the cancellation of all parameters not related toaffinity and is equal to the dissociation constant K_(d). (See,generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). Anantibody of the present disclosure is said to specifically bind to EGFR,when the binding constant (K_(d)) is ≤1 μM, for example, in someembodiments ≤100 nM, in some embodiments 10 nM, and in some embodiments≤100 μM to about 1 μM, as measured by assays such as radioligand bindingassays or similar assays known to those skilled in the art.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide that it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence. Polynucleotides inaccordance with the disclosure include the nucleic acid moleculesencoding the heavy chain immunoglobulin molecules shown herein, andnucleic acid molecules encoding the light chain immunoglobulin moleculesshown herein.

The term “isolated protein” referred to herein means a protein of cDNA,recombinant RNA, or synthetic origin or some combination thereof, whichby virtue of its origin, or source of derivation, the “isolated protein”(1) is not associated with proteins found in nature, (2) is free ofother proteins from the same source, e.g., free of murine proteins, (3)is expressed by a cell from a different species, or (4) does not occurin nature.

The term “polypeptide” is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein fragments, and analogs are species of the polypeptidegenus. Polypeptides in accordance with the disclosure comprise the heavychain immunoglobulin molecules shown herein, and the light chainimmunoglobulin molecules shown herein, as well as antibody moleculesformed by combinations comprising the heavy chain immunoglobulinmolecules with light chain immunoglobulin molecules, such as kappa lightchain immunoglobulin molecules, and vice versa, as well as fragments andanalogs thereof.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andthat has not been intentionally modified by man in the laboratory orotherwise is naturally-occurring.

The term “operably linked” as used herein refers to positions ofcomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

The term “control sequence” as used herein refers to polynucleotidesequences that are necessary to effect the expression and processing ofcoding sequences to which they are ligated. The nature of such controlsequences differs depending upon the host organism in prokaryotes, suchcontrol sequences generally include promoter, ribosomal binding site,and transcription termination sequence in eukaryotes, generally, suchcontrol sequences include promoters and transcription terminationsequence. The term “control sequences” is intended to include, at aminimum, all components whose presence is essential for expression andprocessing, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences. The term “polynucleotide” as referred to herein meansnucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes single and double stranded forms of DNA.

The term “oligonucleotide” referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset generally comprising alength of 200 bases or fewer. In some embodiments, oligonucleotides are10 to 60 bases in length, for example, in some embodiments, 12, 13, 14,15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides areusually single stranded, e.g., for probes, although oligonucleotides maybe double stranded, e.g., for use in the construction of a gene mutant.Oligonucleotides of the disclosure are either sense or antisenseoligonucleotides.

The term “naturally occurring nucleotides” referred to herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes oligonucleotide linkages such asphosphorothioate, phosphorodithioate, phosphoroselerloate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoronmidate, and the like. See e.g., LaPlanche et al. Nucl. AcidsRes. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984),Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. Anti CancerDrug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;Uhlmann and Peyman Chemical Reviews 90:543 (1990). An oligonucleotidecan include a label for detection, if desired.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland? Mass. (1991)). Stereoisomers (e.g., D-amino acids) of thetwenty conventional amino acids, unnatural amino acids such as α-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and otherunconventional amino acids may also be suitable components forpolypeptides of the present disclosure. Examples of unconventional aminoacids include: 4 hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,σ-N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, theleft-hand direction is the amino terminal direction and the right-handdirection is the carboxy-terminal direction, in accordance with standardusage and convention.

Similarly, unless specified otherwise, the left-hand end ofsingle-stranded polynucleotide sequences is the 5′ end the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction sequenceregions on the DNA strand having the same sequence as the RNA and thatare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”, sequence regions on the DNA strand having the same sequenceas the RNA and that are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, for example, in some embodiments, at least 90 percentsequence identity, in some embodiments, at least 95 percent sequenceidentity, and in some embodiments, at least 99 percent sequenceidentity.

In some embodiments, residue positions that are not identical differ byconservative amino acid substitutions.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present disclosure, providing that the variations inthe amino acid sequence maintain at least 75%, for example, in someembodiments, at least 80%, 90%, 95%, and in some embodiments 99%. Inparticular, conservative amino acid replacements are contemplated.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains. Genetically encodedamino acids are generally divided into families: (1) acidic amino acidsare aspartate, glutamate; (2) basic amino acids are lysine, arginine,histidine; (3) non-polar amino acids are alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan, and (4)uncharged polar amino acids are glycine, asparagine, glutamine,cysteine, serine, threonine, tyrosine. The hydrophilic amino acidsinclude arginine, asparagine, aspartate, glutamine, glutamate,histidine, lysine, serine, and threonine. The hydrophobic amino acidsinclude alanine, cysteine, isoleucine, leucine, methionine,phenylalanine, proline, tryptophan, tyrosine and valine. Other familiesof amino acids include (i) serine and threonine, which are thealiphatic-hydroxy family; (ii) asparagine and glutamine, which are theamide containing family; (iii) alanine, valine, leucine and isoleucine,which are the aliphatic family; and (iv) phenylalanine, tryptophan, andtyrosine, which are the aromatic family. For example, it is reasonableto expect that an isolated replacement of a leucine with an isoleucineor valine, an aspartate with a glutamate, a threonine with a serine, ora similar replacement of an amino acid with a structurally related aminoacid will not have a major effect on the binding or properties of theresulting molecule, especially if the replacement does not involve anamino acid within a framework site. Whether an amino acid change resultsin a functional peptide can readily be determined by assaying thespecific activity of the polypeptide derivative. Assays are described indetail herein. Fragments or analogs of antibodies or immunoglobulinmolecules can be readily prepared by those of ordinary skill in the art.In some embodiments, amino- and carboxy-termini of fragments or analogsoccur near boundaries of functional domains. Structural and functionaldomains can be identified by comparison of the nucleotide and/or aminoacid sequence data to public or proprietary sequence databases.Computerized comparison methods are used to identify sequence motifs orpredicted protein conformation domains that occur in other proteins ofknown structure and/or function. Methods to identify protein sequencesthat fold into a known three-dimensional structure are known. Bowie etal. Science 253:164 (1991). Thus, the foregoing examples demonstratethat those of skill in the art can recognize sequence motifs andstructural conformations that may be used to define structural andfunctional domains in accordance with the disclosure.

In some embodiments, amino acid substitutions are those that: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions (forexample, conservative amino acid substitutions) may be made in thenaturally-occurring sequence (for example, in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et at. Nature 354:105 (1991).

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino terminal and/or carboxy-terminal deletion and/or oneor more internal deletion(s), but where the remaining amino acidsequence is identical to the corresponding positions in thenaturally-occurring sequence deduced, for example, from a full lengthcDNA sequence. Fragments typically are at least 5, 6, 8 or 10 aminoacids long, for example, in some embodiments, at least 14 amino acidslong, in some embodiments, at least 20 amino acids long, usually atleast 50 amino acids long, and in some embodiments, at least 70 aminoacids long. The term “analog” as used herein refers to polypeptides thatare comprised of a segment of at least 25 amino acids that hassubstantial identity to a portion of a deduced amino acid sequence andthat has specific binding to EGFR, under suitable binding conditions.Typically, polypeptide analogs comprise a conservative amino acidsubstitution (or addition or deletion) with respect to thenaturally-occurring sequence. Analogs typically are at least 20 aminoacids long, for example, in some embodiments, at least 50 amino acidslong or longer, and can often be as long as a full-lengthnaturally-occurring polypeptide.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). In certain situations, the label or marker canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., a fluorophore, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance. The term “pharmaceutical agent ordrug” as used herein refers to a chemical compound or compositioncapable of inducing a desired therapeutic effect when properlyadministered to a patient.

As used herein, a composition refers to any mixture of two or moreproducts, substances, or compounds, including cells. It may be asolution, a suspension, liquid, powder, a paste, aqueous, non-aqueous orany combination thereof.

The term “pharmaceutical composition” refers to a composition suitablefor pharmaceutical use in a mammalian subject, often a human. Apharmaceutical composition typically comprises an effective amount of anactive agent (e.g., multispecific polypeptide construct) and a carrier,excipient, or diluent. The carrier, excipient, or diluent is typically apharmaceutically acceptable carrier, excipient or diluent, respectively.

The terms “treating,” “treatment,” or “therapy” of a disease or disorderas used herein mean slowing, stopping or reversing the disease ordisorders progression, as evidenced by decreasing, cessation orelimination of either clinical or diagnostic symptoms, by administrationof a pharmaceutical composition of the disclosure either alone or incombination with another compound as described herein. “Treating,”“treatment,” or “therapy” also means a decrease in the severity ofsymptoms in an acute or chronic disease or disorder or a decrease in therelapse rate. As used herein in the context of cancer, the terms“treatment” or, “inhibit,” “inhibiting” or “inhibition” of cancer refersto at least one of: a statistically significant decrease in the rate oftumor growth, a cessation of tumor growth, or a reduction in the size,mass, metabolic activity, or volume of the tumor, as measured bystandard criteria such as, but not limited to, the Response EvaluationCriteria for Solid Tumors (RECIST), or a statistically significantincrease in progression free survival (PFS) or overall survival (OS).“Preventing,” “prophylaxis,” or “prevention” of a disease or disorderrefers to administration of a pharmaceutical composition, either aloneor in combination with another compound, to prevent the occurrence oronset of a disease or disorder or some or all of the symptoms of adisease or disorder or to lessen the likelihood of the onset of adisease or disorder.

The terms “effective amount” or “therapeutically effective amount” referto a quantity and/or concentration of a composition that whenadministered into a patient either alone (i.e., as a monotherapy) or incombination with additional therapeutic agents, yields a statisticallysignificant decrease in disease progression as, for example, byameliorating or eliminating symptoms and/or the cause of the disease. Aneffective amount may be an amount that relieves, lessens, or alleviatesat least one symptom or biological response or effect associated with adisease or disorder, prevents progression of the disease or disorder, orimproves physical functioning of the patient.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present.

Generally, a substantially pure composition will comprise more thanabout 80 percent of all macromolecular species present in thecomposition, for example, in some embodiments, more than about 85%, 90%,95%, and 99%. In some embodiments, the object species is purified toessential homogeneity (contaminant species cannot be detected in thecomposition by conventional detection methods) wherein the compositionconsists essentially of a single macromolecular species.

The term patient includes human and veterinary subjects.

Other chemistry terms herein are used according to conventional usage inthe art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

II. MULTISPECIFIC POLYPEPTIDE CONSTRUCTS

Provided herein is a multispecific polypeptide construct containing afirst component containing an immunoglobulin Fc region and a secondcomponent comprising a CD3-binding region, wherein the first and secondcomponents are coupled by a linker, wherein the Fc region is positionedN-terminal to the CD3-binding region; and one or both of the first andsecond components comprises an antigen binding domain that binds a tumorassociated antigen (TAA). In some embodiments, the linker is anon-cleavable linker. In some embodiments, the linker does not contain asubstrate recognition that is specifically recognized for cleavage by aprotease.

In some embodiments, the multispecific polypeptide construct contains inorder, from N-terminus to C-terminus: an immunoglobulin Fc region; alinker; a CD3 binding region that binds CD3 (CD3ε); and an antigenbinding domain that binds a tumor-associated antigen (TAA). In someembodiments, the multispecific polypeptide construct contains in order,from N-terminus to C-terminus: an antigen binding domain that binds to atumor-associated antigen (TAA); an immunoglobulin Fc region; a linker;and a CD3 binding region that binds CD3 (CD3ε). In some embodiments, themultispecific polypeptide construct contains at least a first antigenbinding domain that binds a TAA and a second antigen binding domain thatbinds a TAA. In some embodiments, the multispecific polypeptideconstruct contains, in order, from N-terminus to C-terminus: a firstantigen binding domain that binds to a tumor-associated antigen (TAA);an immunoglobulin Fc region; a linker; a CD3 binding region that bindsCD3 (CD3ε); and a second antigen binding domain that binds atumor-associated antigen (TAA).

Each of the components of the multispecific polypeptide constructs ofthe disclosure is described in more detail below.

1. Anti-CD3 Binding Domains:

The multispecific polypeptide constructs of the disclosure include oneor more copies of an anti-CD3 binding domain. The anti-CD3 bindingdomains of the disclosure activate T cells via engagement of CD3ε on theT cells. The anti-CD3 binding domains of the disclosure agonize,stimulate, activate, and/or otherwise augment CD3-mediated T cellactivation. Biological activities of CD3 include, for example, T cellactivation and other signaling through interaction between CD3 and theantigen-binding subunits of the T-Cell Receptor (TCR). For example, theanti-CD3 binding domains of the disclosure completely or partiallyactivate T cells via engagement of CD3ε on T cells by partially orcompletely modulating, e.g., agonizing, stimulating, activating orotherwise augmenting CD3-mediated T cell activation.

In preferred embodiments, the anti-CD3 binding domains of the disclosurespecifically bind the epsilon chain of CD3, also known as CD3ε. Theanti-CD3ε binding domains of the disclosure activate T cells viaengagement of CD3ε on the T cells. The anti-CD3ε binding domains of thedisclosure include monoclonal antibodies, such as, for example,mammalian monoclonal antibodies, primate monoclonal antibodies, fullyhuman monoclonal antibodies, as well as humanized monoclonal antibodiesand chimeric antibodies, as well as antigen-binding fragments thereof.In some embodiments, the anti-CD3ε binding domain includes one or morecopies of an antibody or an antigen-binding fragment thereof.

In some embodiments, the anti-CD3ε binding domain includes a combinationof a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence,wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, andthe VH CDR3 sequence is selected from a VH CDR1 sequence that includesat least the amino acid sequence TYAMN (SEQ ID NO: 16); a VH CD2sequence that includes at least the amino acid sequenceRIRSKYNNYATYYADSVKD (SEQ ID NO: 17); and a VH CDR3 sequence thatincludes at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO:18). In some embodiments, the anti-CD3ε binding domain includes acombination of a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3sequence, wherein at least one of the VH CDR1 sequence, the VH CDR2sequence, and the VH CDR3 sequence is selected from a VH CDR1 sequencethat includes at least the amino acid sequence GFTFNTYAMN (SEQ ID NO:211); a VH CDR2 sequence that includes at least the amino acid sequenceRIRSKYNNYATY (SEQ ID NO: 212); and a VH CDR3 sequence that includes atleast the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 18).

In some embodiments, the anti-CD3ε binding domain includes a combinationof a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence,wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, andthe VL CDR3 sequence is selected from a VL CDR1 sequence that includesat least the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 19); a VLCDR2 sequence that includes at least the amino acid sequence GTNKRAP(SEQ ID NO: 20); and a VL CDR3 sequence that includes at least the aminoacid sequence ALWYSNLWV (SEQ ID NO: 21).

In some embodiments, the anti-CD3ε binding domain includes a VH CDR1sequence that includes at least the amino acid sequence TYAMN (SEQ IDNO: 16); a VH CDR2 sequence that includes at least the amino acidsequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 17); a VH CDR3 sequence thatincludes at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO:18), a VL CDR1 sequence that includes at least the amino acid sequenceRSSTGAVTTSNYAN (SEQ ID NO: 19); a VL CDR2 sequence that includes atleast the amino acid sequence GTNKRAP (SEQ ID NO: 20); and a VL CDR3sequence that includes at least the amino acid sequence ALWYSNLWV (SEQID NO: 21).

In some embodiments, the anti-CD3ε binding domain includes a VH CDR1sequence that includes at least the amino acid sequence GFTFNTYAMN (SEQID NO: 211); a VH CDR2 sequence that includes at least the amino acidsequence RIRSKYNNYATY (SEQ ID NO: 212); a VH CDR3 sequence that includesat least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 18), a VLCDR1 sequence that includes at least the amino acid sequenceRSSTGAVTTSNYAN (SEQ ID NO: 19); a VL CDR2 sequence that includes atleast the amino acid sequence GTNKRAP (SEQ ID NO: 20); and a VL CDR3sequence that includes at least the amino acid sequence ALWYSNLWV (SEQID NO: 21).

In some embodiments, the anti-CD3ε binding domain includes a VH CDR1sequence that includes at least the amino acid sequence GFTFNTYAMN (SEQID NO: 211); a VH CDR2 sequence that includes at least the amino acidsequence RIRSKYNNYATY (SEQ ID NO: 212); a VH CDR3 sequence that includesat least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 18), a VLCDR1 sequence that includes at least the amino acid sequenceGSSTGAVITSNYAN (SEQ ID NO: 229); a VL CDR2 sequence that includes atleast the amino acid sequence GTNKRAP (SEQ ID NO: 230); and a VL CDR3sequence that includes at least the amino acid sequence ALWYSNHWV (SEQID NO: 225).

In some embodiments, the anti-CD3ε binding domain includes a combinationof a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence,wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, andthe VH CDR3 sequence is selected from a VH CDR1 sequence that includes asequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the amino acid sequence TYAMN (SEQ ID NO: 16);a VH CDR2 sequence that includes a sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the aminoacid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 17); and a VH CDR3sequence that includes a sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acidsequence HGNFGNSYVSWFAY (SEQ ID NO: 18).

In some embodiments, the anti-CD3ε binding domain includes a combinationof a VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence,wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence, andthe VH CDR3 sequence is selected from a VH CDR1 sequence that includes asequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the amino acid sequence GFTFNTYAMN (SEQ ID NO:211); a VH CDR2 sequence that includes a sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to theamino acid sequence RIRSKYNNYATY (SEQ ID NO: 212); and a VH CDR3sequence that includes a sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acidsequence HGNFGNSYVSWFAY (SEQ ID NO: 18).

In some embodiments, the anti-CD3ε binding domain includes a combinationof a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence,wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence, andthe VL CDR3 sequence is selected from a VL CDR1 sequence that includes asequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the amino acid sequence RSSTGAVTTSNYAN (SEQ IDNO: 19); a VL CDR2 sequence that includes a sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical tothe amino acid sequence GTNKRAP (SEQ ID NO: 20); and a VL CDR3 sequencethat includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the amino acid sequenceALWYSNLWV (SEQ ID NO: 21).

In some embodiments, the anti-CD3ε binding domain includes a VH CDR1sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the amino acid sequence TYAMN (SEQ ID NO: 16);a VH CD2 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to the amino acid sequenceRIRSKYNNYATYYADSVKD (SEQ ID NO: 17); a VH CDR3 sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical tothe amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 18), a VL CDR1sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the amino acid sequence RSSTGAVTTSNYAN (SEQ IDNO: 19); a VL CDR2 sequence that is at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequenceGTNKRAP (SEQ ID NO: 20); and a VL CDR3 sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to theamino acid sequence ALWYSNLWV (SEQ ID NO: 21).

In some embodiments, the anti-CD3ε binding domain includes a VH CDR1sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the amino acid sequence GFTFNTYAMN (SEQ ID NO:211); a VH CDR2 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the amino acid sequenceRIRSKYNNYATY (SEQ ID NO: 212); a VH CDR3 sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to theamino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 18), a VL CDR1 sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO:19); a VL CDR2 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the amino acid sequence GTNKRAP(SEQ ID NO: 20); and a VL CDR3 sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acidsequence ALWYSNLWV (SEQ ID NO: 21).

In some embodiments, the anti-CD3ε binding domain includes a VH CDR1sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the amino acid sequence GFTFNTYAMN (SEQ ID NO:211); a VH CDR2 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to amino acid sequence RIRSKYNNYATY(SEQ ID NO: 212); a VH CDR3 sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acidsequence HGNFGNSYVSWFAY (SEQ ID NO: 18), a VL CDR1 sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence GSSTGAVTTSNYAN (SEQ ID NO: 229); a VL CDR2sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the amino acid sequence GTNKRAP (SEQ ID NO:230); and a VL CDR3 sequence that is at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequenceALWYSNHWV (SEQ ID NO: 225).

In some embodiments, the anti-CD3ε binding domain includes a VH CDR1sequence that includes at least the amino acid sequence GFTSTYAMN (SEQID NO: 227); a VH CDR2 sequence that includes at least the amino acidsequence RIRSKYNNYATY (SEQ ID NO: 228); a VH CDR3 sequence that includesat least the amino acid sequence HGNFGDSYVSWFAY (SEQ ID NO: 224), a VLCDR1 sequence that includes at least the amino acid sequenceGSSTGAVTTSNYAN (SEQ ID NO: 229); a VL CDR2 sequence that includes atleast the amino acid sequence GTNKRAP (SEQ ID NO: 230); and a VL CDR3sequence that includes at least the amino acid sequence ALWYSNHWV (SEQID NO: 225).

In some embodiments, the anti-CD3ε binding domain includes a VH CDR1sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the amino acid sequence GFTFSTYAMN (SEQ ID NO:227); a VH CDR2 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the amino acid sequenceRIRSKYNNYATY (SEQ ID NO: 228); a VH CDR3 sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to theamino acid sequence HGNFGDSYVTSWFAY (SEQ ID NO: 224), a VL CDR1 sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the amino acid sequence GSSTGAVTTSNYAN (SEQ ID NO:229); a VL CDR2 sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the amino acid sequence GTNKRAP(SEQ ID NO: 230); and a VL CDR3 sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acidsequence ALWYSNHWV (SEQ ID NO: 225).

In some embodiments, the anti-CD3ε binding domain includes a CDR3 thatincludes at least amino acids VLWYSNRWV (SEQ ID NO:226). In someembodiments, the anti-CD3ε binding domain includes a CDR3 that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acids VLWYSNRWV (SEQ ID NO:226).

In some embodiments, the anti-CD3ε binding domain includes one or morecopies of an antibody or an antigen-binding fragment thereof selectedfrom the group consisting of a Fab fragment, a F(ab)₂ fragment, an Fvfragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody,and a single domain light chain antibody. In some embodiments, theanti-CD3 binding domain includes an Fv antibody fragment that binds CD3ε(referred to herein as an anti-CD3ε Fv fragment). In some embodiments,the anti-CD3ε Fv antibody fragment is a disulfide stabilized anti-CD3binding Fv fragment (dsFv). In some embodiments, the anti-CD3 bindingdomain is monovalent for binding CD3.

In some embodiments, the CD3 binding region is not a single chainantibody. For example, in some aspects, the CD3 binding region is not asingle chain variable fragment (scFv).

In some embodiments, the CD3 binding region is an Fv antibody fragmentcontaining a variable heavy chain (Hv, also called VH) and variablelight chain (Lv, also called VL), such as any as described. In aspectsof such embodiments, the immunoglobulin Fc region is a heterodimeric Fcregion containing two different Fc polypeptides capable of heterodimericassociation between both polypeptides of the Fc heterodimer, such as anyas described in Section 11.2. In such embodiments, the variable heavychain (VH) and variable light chain (VL) of the CD3 binding region arelinked on opposite chains of the heterodimeric Fc.

In some embodiments, the anti-CD3ε Fv antibody fragment includes anamino acid sequence selected from the group of SEQ ID NO: 32-81. In someembodiments, the anti-CD3ε Fv antibody fragment includes an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 32-81. In some embodiments, the anti-CD3ε Fvantibody fragment includes a combination of an amino acid sequenceselected from the group of SEQ ID NO: 32-62 and an amino acid sequenceselected from the group consisting of SEQ ID NO: 63-81. In someembodiments, the anti-CD3ε Fv antibody fragment includes a combinationof an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 32-62 and an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 63-81 an amino acid sequence.

In some embodiments, the anti-CD3ε binding domain thereof includes acombination of a heavy chain variable region amino acid sequence and alight chain variable region amino acid sequence comprising an amino acidsequence selected from the group of SEQ ID NO: 32-81. In someembodiments, the anti-CD3ε binding domain thereof includes a combinationof a heavy chain variable region amino acid sequence selected from thegroup of SEQ ID NO: 32-62 and a light chain variable region amino acidsequence comprising an amino acid sequence selected from the group ofSEQ ID NO: 63-81.

In some embodiments, the anti-CD3ε binding domain thereof is an Fvfragment that includes a combination of heavy chain variable amino acidsequence and a light chain variable amino acid sequence. In someembodiments, the anti-CD3ε binding domain thereof is an Fv fragment thatincludes a combination of heavy chain variable amino acid sequence and alight chain variable amino acid sequence comprising an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 14, 15, 32-81, 191, 196-200, 211, and 212. Insome embodiments, the anti-CD3ε binding domain thereof is an Fv fragmentthat includes a combination of heavy chain variable amino acid sequenceand a light chain variable amino acid sequence comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 14, 15,32-81, 191, 196-200, 211, and 212. In some embodiments, the anti-CD3εbinding domain thereof is an Fv fragment that includes a combination ofheavy chain variable amino acid sequence selected from the group of SEQID NO: 14, 32-62, 196-198, and 211 and light chain variable amino acidsequence selected from the group consisting of SEQ ID NO: 15, 63-81,191, 199, 200, and 212. In some embodiments, the anti-CD3ε bindingdomain thereof is an Fv fragment that includes a combination of heavychain variable amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequenceselected from the group consisting of SEQ ID NO: 14, 32-62, 196-198, and211 and a light chain variable amino acid sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to anamino acid sequence selected from the group consisting of SEQ ID NO: 15,63-81, 191, 199, 200, and 212.

In some embodiments, the anti-CD3ε binding domain thereof includes acombination of a heavy chain variable region amino acid sequence and alight chain variable region amino acid sequence comprising an amino acidsequence selected from the group of SEQ ID NO: 32-81, 191, 196-200, 211,and 212. In some embodiments, the anti-CD3ε binding domain thereofincludes a combination of a heavy chain variable region amino acidsequence selected from the group of SEQ ID NO: 32-62, 196-198, and 211and a light chain variable region amino acid sequence comprising anamino acid sequence selected from the group of SEQ ID NO: 63-81, 191,199, 200, and 212.

In some embodiments, the anti-CD3ε Fv antibody fragment includes acombination of an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acidsequence selected from the group consisting of SEQ ID NO: 14, 32-43,45-47, 48, 196 and 211 and an amino acid sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to anamino acid sequence selected from the group consisting of SEQ ID NO: 15,63, 65-71, 73, 75, 77, and 199. In some embodiments, the anti-CD3ε Fvantibody fragment includes a combination of an amino acid sequenceselected from the group of SEQ ID NO: 14, 32-43, 45-47, 48, 196 and 211and an amino acid sequence selected from the group consisting of SEQ IDNO: 15, 63, 65-71, 73, 75, 77, and 199.

In some embodiments, the anti-CD3ε binding domain thereof includes avariable heavy chain (VH) comprising an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence of SEQ ID NO: 14. In some embodiments, theanti-CD3ε binding domain includes a variable light chain (VL) comprisingan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQID NO: 15. In some embodiments, the anti-CD3ε binding domain thereofincludes a variable heavy chain (VH) comprising an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the amino acid sequence of SEQ ID NO: 14 and avariable light chain (VL) comprising an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence of SEQ ID NO: 15. In some embodiments, theanti-CD3ε binding domain thereof includes a variable heavy chain (VH)comprising the amino acid sequence of SEQ ID NO: 14. In someembodiments, the anti-CD3ε binding domain includes a variable lightchain (VL) comprising the amino acid sequence of SEQ ID NO: 15. In someembodiments, the anti-CD3ε binding domain thereof includes a variableheavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 14 anda variable light chain (VL) comprising the amino acid sequence of SEQ IDNO: 15.

In some embodiments, the anti-CD3ε binding domain thereof includes avariable heavy chain (VH) comprising an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence of SEQ ID NO: 196. In some embodiments, theanti-CD3ε binding domain includes a variable light chain (VL) comprisingan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQID NO: 199. In some embodiments, the anti-CD3ε binding domain thereofincludes a variable heavy chain (VH) comprising an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the amino acid sequence of SEQ ID NO: 196 and avariable light chain (VL) comprising an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence of SEQ ID NO: 199. In some embodiments, theanti-CD3ε binding domain thereof includes a variable heavy chain (VH)comprising the amino acid sequence of SEQ ID NO: 196. In someembodiments, the anti-CD3ε binding domain includes a variable lightchain (VL) comprising the amino acid sequence of SEQ ID NO: 199. In someembodiments, the anti-CD3ε binding domain thereof includes a variableheavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 196and a variable light chain (VL) comprising the amino acid sequence ofSEQ ID NO: 199.

In particular embodiments, the Fv is a disulfide stabilized Fv fragment(dsFv) in which the V_(H)-V_(L) heterodimer is stabilized by aninterchain disulfide bond. In some embodiments, the interchain disulfidebond is engineered by mutation of position in framework positions of theVH and/or VL chain. In some embodiments, the disulfide stabilizedanti-CD3 Fv comprises an anti-CD3 VH with the mutation 44 to Cys and ananti-CD3 VL with the mutation 100 to Cys by Kabat numbering. Forexample, in some embodiments, the VH chain contains the mutation G44Cand the VL chain contains the mutation G100C, each by kabat numbering.In some embodiments, the disulfide stabilized anti-CD3 Fv comprises ananti-CD3 VH with the mutation at position 105 to Cys and an anti-CD3 VLwith the mutation position 43 to Cys by Kabat numbering.

In some embodiments, the anti-CD3ε Fv antibody fragment includes acombination of an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acidsequence selected from the group consisting of SEQ ID NO: 44, 49-62, 197and 198 and an amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequenceselected from the group consisting of SEQ ID NO: 64, 72, 74, 76, 78-81,191, 200 and 212. In some of any such embodiments, the anti-CD3 Fv is adsFv that has a VH chain containing the mutation G44C and a VL chaincontaining the mutation G100C, each by kabat numbering. In someembodiments, the anti-CD3ε Fv antibody fragment includes a combinationof an amino acid sequence selected from the group of SEQ ID NO: 44,49-62, 197 and 198 and an amino acid sequence selected from the groupconsisting of SEQ ID NO: 64, 72, 74, 76, 78-81, 191, 200 and 212.

In some embodiments, the anti-CD3ε binding domain thereof includes avariable heavy chain (VH) comprising an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence of SEQ ID NO: 44. In some embodiments, theanti-CD3ε binding domain includes a variable light chain (VL) comprisingan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQID NO: 72. In some embodiments, the anti-CD3ε binding domain thereofincludes a variable heavy chain (VH) comprising an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the amino acid sequence of SEQ ID NO: 44 and avariable light chain (VL) comprising an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence of SEQ ID NO: 72. In some of any suchembodiments, the anti-CD3 Fv is a dsFv that has a VH chain containingthe mutation G44C and a VL chain containing the mutation G100C, each bykabat numbering. In some embodiments, the anti-CD3ε binding domainthereof includes a variable heavy chain (VH) comprising the amino acidsequence of SEQ ID NO: 44. In some embodiments, the anti-CD3ε bindingdomain includes a variable light chain (VL) comprising the amino acidsequence of SEQ ID NO: 72. In some embodiments, the anti-CD3ε bindingdomain thereof includes a variable heavy chain (VH) comprising the aminoacid sequence of SEQ ID NO: 44 and a variable light chain (VL)comprising the amino acid sequence of SEQ ID NO: 72.

In some embodiments, the anti-CD3ε binding domain thereof includes avariable heavy chain (VH) comprising an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence of SEQ ID NO: 198. In some embodiments, theanti-CD3ε binding domain includes a variable light chain (VL) comprisingan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQID NO: 200. In some embodiments, the anti-CD3ε binding domain thereofincludes a variable heavy chain (VH) comprising an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the amino acid sequence of SEQ ID NO: 198 and avariable light chain (VL) comprising an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence of SEQ ID NO: 200. In some of any suchembodiments, the anti-CD3 Fv is a dsFv that has a VH chain containingthe mutation G44C and a VL chain containing the mutation G100C, each bykabat numbering. In some embodiments, the anti-CD3ε binding domainthereof includes a variable heavy chain (VH) comprising the amino acidsequence of SEQ ID NO: 198. In some embodiments, the anti-CD3ε bindingdomain includes a variable light chain (VL) comprising the amino acidsequence of SEQ ID NO: 200. In some embodiments, the anti-CD3ε bindingdomain thereof includes a variable heavy chain (VH) comprising the aminoacid sequence of SEQ ID NO: 198 and a variable light chain (VL)comprising the amino acid sequence of SEQ ID NO: 200.

In some embodiments, the anti-CD3ε binding domain thereof includes avariable heavy chain (VH) comprising an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence of SEQ ID NO: 197. In some embodiments, theanti-CD3ε binding domain includes a variable light chain (VL) comprisingan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQID NO: 200. In some embodiments, the anti-CD3ε binding domain thereofincludes a variable heavy chain (VH) comprising an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the amino acid sequence of SEQ ID NO: 197 and avariable light chain (VL) comprising an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence of SEQ ID NO: 200. In some of any suchembodiments, the anti-CD3 Fv is a dsFv that has a VH chain containingthe mutation G44C and a VL chain containing the mutation G100C, each bykabat numbering. In some embodiments, the anti-CD3ε binding domainthereof includes a variable heavy chain (VH) comprising the amino acidsequence of SEQ ID NO: 197. In some embodiments, the anti-CD3ε bindingdomain includes a variable light chain (VL) comprising the amino acidsequence of SEQ ID NO: 200. In some embodiments, the anti-CD3ε bindingdomain thereof includes a variable heavy chain (VH) comprising the aminoacid sequence of SEQ ID NO: 197 and a variable light chain (VL)comprising the amino acid sequence of SEQ ID NO: 200.

2. Immunoglobulin Fc Polypeptides:

The first component of the multispecific polypeptide constructs of thedisclosure includes an immunoglobulin Fc region. In some embodiments,the immunoglobulin Fc region is an IgG isotype selected from the groupconsisting of IgG1 isotype, IgG2 isotype, IgG3 isotype, and IgG4subclass. In some embodiments, the Fc region is a human Fc. In someembodiments, the immunoglobulin Fc region is a polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:1-6. In some embodiments, the immunoglobulin Fc region contains an Fcchain that is an immunologically active fragment of any of SEQ ID Nos:1-6. In some embodiments, the immunoglobulin Fc region contains an Fcpolypeptide chain that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to theamino acid sequence of any of SEQ ID NOs: 1-6 or an immunologicallyactive fragment thereof.

In some embodiments, the multispecific polypeptide construct is a dimerformed by polypeptides, each containing an Fc. In some specificembodiments, identical or substantially identical polypeptides will bedimerized to create a homodimer. In some embodiments, the dimer is ahomodimer in which the two polypeptides of the multispecific polypeptideconstruct are the same. In other cases, the Fc region is formed by Fcdomains that are mutated or modified to promote heterodimerization inwhich different polypeptides can be dimerized to yield a heterodimer.Thus, in some embodiments, the dimer is a heterodimer in which twopolypeptide chains of the multispecific polypeptide construct aredifferent. Exemplary modifications to promote heterodimerization areknown, including any as described below.

In general, the Fc region is responsible for effector functions, such ascomplement-dependent cytotoxicity (CDC) and antibody-dependent cellcytotoxicity (ADCC), in addition to the antigen-binding capacity, whichis the main function of immunoglobulins. Additionally, the FcRn sequencepresent in the Fc region plays the role of regulating the IgG level inserum by increasing the in vivo half-life by conjugation to an in vivoFcRn receptor. In some embodiments, such functions can be altered, suchas reduced or enhanced, in an Fc for use with the provided multispecificpolypeptide constructs.

In some embodiments, the Fc region of the provided multispecificpolypeptide constructs exhibit one or more effector functions. In somecases, the Fc region is capable of providing Fc-mediated effectorfunctions, such as for example, ADCC (e.g., release of granzyme B by NKcells), ADCP, and/or CDC. Thus, in some embodiments in which themultispecific polypeptide constructs contain a cleavable linker,cleavage of the linker can produce two components that each havebiological activity: the CD3-binding region that is able to bind andengage CD3 on a T cell and the Fc region linked to the TAA-antigenbinding domain that can exhibit target-specific effector function. Inparticular embodiments provided herein, the multispecific polypeptideconstructs contain a non-cleavable linker and may, in some aspects, notexhibit an independent Fc-mediated effector function.

In some embodiments, the Fc region includes an Fc polypeptide that ismutated or modified to alter one or more effector functions. Variousexamples of mutations to Fc polypeptides to alter, such as reduce,effector function are known, including any as described below. In someembodiments, reference to amino acid substitutions in an Fc region is byEU numbering by Kabat (also called Kabat numbering) unless describedwith reference to a specific SEQ ID NO. EU numbering is known and isaccording to the most recently updated IMGT Scientific Chart (IMGT®, theinternational ImMunoGeneTics information System®,http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html(created: 17 May 2001, last updated: 10 Jan. 2013) and the EU index asreported in Kabat, E. A. et al. Sequences of Proteins of Immunologicalinterest. 5th ed. US Department of Health and Human Services, NIHpublication No. 91-3242 (1991).

In some embodiments, provided multispecific polypeptide constructs thatcontain an Fc region that exhibits reduced effector functions, may be adesirable candidate for applications in which constrained CD3 binding isdesired yet certain effector functions (such as CDC and ADCC) areunnecessary or deleterious. In vitro and/or in vivo cytotoxicity assayscan be conducted to confirm the reduction/depletion of CDC and/or ADCCactivities. For example, Fc receptor (FcR) binding assays can beconducted to ensure that the multispecific polypeptide constructs and/orcleaved components thereof lack FcγR binding (hence likely lacking ADCCactivity), but retains FcRn binding ability. The primary cells formediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII. Non-limiting examples of in vitroassays to assess ADCC activity of a molecule of interest is described inU.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'lAcad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'lAcad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (seeBruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).Alternatively, non-radioactive assay methods may be employed (see, forexample, ACTI™ non-radioactive cytotoxicity assay for flow cytometry(CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96™non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in an animal model such as that disclosed in Clynes etal. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays mayalso be carried out to confirm that the multispecific polypeptideconstruct or cleaved components thereof is unable to bind C1q and hencelacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO2006/029879 and WO 2005/100402. To assess complement activation, a CDCassay may be performed (see, for example, Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood103:2738-2743 (2004)). FcRn binding and in vivo clearance/half-lifedeterminations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769(2006)).

In some embodiments, the immunoglobulin Fc region or immunologicallyactive fragment thereof is an IgG isotype. For example, theimmunoglobulin Fc region of the fusion protein is of human IgG1 isotype,having an amino acid sequence:

(SEQ ID NO: 1)

APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAVEWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMHEALHNHYTQK SLSLSPGK

In some embodiments, the immunoglobulin Fc region or immunologicallyactive fragment thereof comprises a human IgG1 polypeptide sequence thatis at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQID NO: 1.

In some embodiments, an IgG1 Fc polypeptide or a variant thereof such asany described below can be made in a G1 ml or G1 m3 allotype. In someembodiments, the Fc region can contain amino acids of the human G1 mlallotype, such as residues containing Asp (D) and Leu (L) at positions356 and 358, e.g. as set forth in SEQ ID NO:1. In some cases, an Fcpolypeptide can contain amino acid substitutions E356D and M358L toreconstitute residues of allotype G1 ml. In other embodiments, the Fcregion can contain amino acids of the human G1 m3 allotype, such asresidues Glu (E) and Met (M) at positions 356 and 358 by EU numbering,e.g. as set forth in SEQ ID NOS: 194 and 195. In some cases, an Fcpolypeptide can contain amino acid substitutions D356E and L358M toreconstitute residues of allotype G1 m3. In some embodiments, the humanIgG1 Fc region is modified to alter antibody-dependent cellularcytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC),e.g., the amino acid modifications described in Natsume et al., 2008Cancer Res, 68(10): 3863-72; Idusogie et al., 2001 J Immunol, 166(4):2571-5; Moore et al., 2010 mAbs, 2(2): 181-189; Lazar et al., 2006 PNAS,103(11): 4005-4010, Shields et al., 2001 JBC, 276(9): 6591-6604;Stavenhagen et al., 2007 Cancer Res, 67(18): 8882-8890; Stavenhagen etal., 2008 Advan. Enzyme Regul., 48: 152-164; Alegre et al, 1992 JImmunol, 148: 3461-3468; Reviewed in Kaneko and Niwa, 2011 Biodrugs,25(1):1-11, the contents of each of which are hereby incorporated byreference in their entireties.

In some embodiments, the Fc region, such as the human IgG1 Fc region ismodified to enhance ADCC activity or CDC activity. Examples of mutationsthat enhance ADCC include modification at Ser239 and Ile332, for exampleSer239Asp and Ile332Glu (S239D, 1332E). Examples of mutations thatenhance CDC include modifications at Lys326 and Glu333. In someembodiments, the Fc region is modified at one or both of thesepositions, for example Lys326Ala and/or Glu333Ala (K326A and E333A)using the Kabat numbering system.

In some embodiments, the human IgG1 Fc region fusion proteins of thepresent disclosure lack or have reduced Fucose attached to the N-linkedglycan-chain at N297. There are numerous ways to prevent fucosylation,including but not limited to production in a FUT8 deficient cell line;addition inhibitors to the mammalian cell culture media, for exampleCastanospermine; and metabolic engineering of the production cell line.In some embodiments, the human IgG1 Fc region is modified at amino acidAsn297 (Boxed, Kabat Numbering) to prevent glycosylation of the fusionprotein, e.g., Asn297Ala (N297A) or Asn297Asp (N297D).

In some embodiments, the Fc region is altered to provide reducedFc-mediated effector functions, such as via reduced Fc receptor binding,e.g. binding to FcγR binding but generally not FcRn binding. In someembodiments, the Fc region of the fusion protein is altered at one ormore of the following positions to reduce Fc receptor binding: Leu 234(L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Ser298 (S298),Asn297 (N297), Asn325 (N325) orAla327 (A327). For example, Leu 234Ala(L234A), Leu235Ala (L235A), Asp265Asn (D265N), Asp270Asn (D270N),Ser298Asn (S298N), Asn297Ala (N297A), Asn325Glu (N325E) orAla327Ser(A327S). In some embodiments, the Fc region of the fusion protein ismodified at amino acid Leu235 (Boxed in SEQ ID NO:1 above, KabatNumbering) to alter Fc receptor interactions, e.g., Leu235Glu (L235E) orLeu235Ala (L235A). In some embodiments, the Fc region of the fusionprotein is modified at amino acid Leu234 (Boxed in SEQ ID NO:1 above,Kabat Numbering) to alter Fc receptor interactions, e.g., Leu234Ala(L234A). In some embodiments, the Fc region of the fusion protein isaltered at both amino acid 234 and 235, e.g., Leu234Ala and Leu235Ala(L234A/L235A) or Leu234Val and Leu235Ala (L234V/L235A). In preferredembodiments, modifications within the Fc region reduce binding toFc-receptor-gamma receptors while have minimal impact on binding to theneonatal Fc receptor (FcRn).

In some embodiments, the human IgG Fc region is modified to enhance FcRnbinding. Examples of Fc mutations that enhance binding to FcRn areMet252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T, T256E, respectively)(Kabat numbering, Dall'Acqua et al 2006, 1 Biol Chem Vol. 281(33)23514-23524), Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et al2010 Nature Biotech, Vol. 28(2) 157-159) (EU index of Kabat et al 1991Sequences of Proteins of Immunological Interest). In some embodiments,the mutated or modified Fc polypeptide includes the following mutations:Met252Tyr and Met428Leu or Met252Tyr and Met428Val (M252Y, M428L, orM252Y, M428V) using the Kabat numbering system.

In some embodiments, the Fc region of the fusion protein is lacking anamino acid at one or more of the following positions to reduce Fcreceptor binding: Glu233 (E233), Leu234 (L234), or Leu235 (L235). Inthese embodiments, Fc deletion of these three amino acids reduces thecomplement protein C1q binding.

(SEQ ID NO: 2) PAPGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPEVKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQDWLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLPPSRDELTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK

In some embodiments, the Fc region is mutated in one or more of thefollowing positions to reduce Fc receptor binding: Glu233 (E233), Leu234(L234), or Leu235 (L235). The one or more mutations can include E233P,L234V and/or L235A.

In some embodiments, the Fc region of the fusion protein is altered atGly236 (boxed in SEQ ID NO:1 above) to reduce Fc receptor binding. Forexample, wherein Gly236 is deleted from the fusion protein. In someembodiments, the human IgG1 Fc region is modified at amino acid Gly236to enhance the interaction with CD32A, e.g., Gly236Ala (G236A).

In particular embodiments, the mutations of the Fc region to reduce Fceffector function, e.g. via reducing Fc receptor binding to FcγR,include mutations from among any of G236R/L328R,E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S267K,E233P/L234V/L235A/G236del/S239K/A327G,E233P/L234V/L235A/G236del/S267K/A327G or E233P/L234V/L235A/G236del.

In some embodiments, the human IgG1 Fc region lacks Lys447 (EU index ofKabat et al 1991 Sequences of Proteins of Immunological Interest).

In some embodiments, the fusion or immunologically active fragmentthereof comprises a human IgG2 polypeptide sequence that is at least50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the immunoglobulin Fc region or immunologicallyactive fragment of the fusion protein is of human IgG2 isotype, havingan amino acid sequence:

(SEQ ID NO: 3) PAPPVAGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFNWYVD

PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDISVEWESNGQPENN YKTTPPMLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK

In some embodiments, the fusion or immunologically active fragmentthereof comprises a human IgG2 polypeptide sequence that is at least50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 3.

In some embodiments, the human IgG2 Fc region is modified at amino acidAsn297 (Boxed, to prevent to glycosylation of the antibody, e.g.,Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the humanIgG2 Fc region lacks Lys447 (EU index of Kabat et al 1991 Sequences ofProteins of Immunological Interest).

In some embodiments, the immunoglobulin Fc region or immunologicallyactive fragment of the fusion protein is of human IgG3 isotype, havingan amino acid sequence:

(SEQ ID NO: 4) PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVQFKWYV

APIEKTISKT KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAVEWESSGQPEN NYNTTPPMLD SDGSFFLYSK LTVDKSRWQQ GNIFSCSVMH

In some embodiments, the antibody or immunologically active fragmentthereof comprises a human IgG3 polypeptide sequence that is at least50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the human IgG3 Fc region is modified at amino acidAsn297 (Boxed, Kabat Numbering) to prevent to glycosylation of theantibody, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In someembodiments, the human IgG3 Fc region is modified at amino acid 435 toextend the half-life, e.g., Arg435His (R435H). In some embodiments, thehuman IgG3 Fc region lacks Lys447 (EU index of Kabat et al 1991Sequences of Proteins of Immunological Interest).

In some embodiments, the immunoglobulin Fc region or immunologicallyactive fragment of the fusion protein is of human IgG4 isotype, havingan amino acid sequence:

(SEQ ID NO: 5)

SSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAVEWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMHEALHNHYTQK SLSLSLGK

In some embodiments, the antibody or immunologically active fragmentthereof comprises a human IgG4 polypeptide sequence that is at least50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 5.

In some embodiments, the immunoglobulin Fc region or immunologicallyactive fragment of the fusion protein is of human IgG4 isotype, havingan amino acid sequence:

(SEQ ID NO: 6) PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSQE DPEVQFNWYV

SSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAVEWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMHEALHNHYTQK SLSLSLGK

In some embodiments, the antibody or immunologically active fragmentthereof comprises a human IgG4 polypeptide sequence that is at least50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 6.

In other embodiments, the human IgG4 Fc region is modified at amino acid235 to alter Fc receptor interactions, e.g., Leu235Glu (L235E). In someembodiments, the human IgG4 Fc region is modified at amino acid Asn297(Boxed, Kabat Numbering) to prevent to glycosylation of the antibody,e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, thehuman IgG4 Fc region lacks Lys447 (EU index of Kabat et al 1991Sequences of Proteins of Immunological Interest).

In some embodiments, the human IgG Fc region is modified to stabilizethe homodimerization at the CH3:CH3 interface by introducing twodisulfide bonds by changing Ser354 to Cys (S354C) and Tyr349 to Cys(Y349C) (S354C/Y349C).

In particular embodiments of multispecific polypeptide constructsprovided herein, the human IgG Fc region is modified to induceheterodimerization. Various methods are known for promotingheterodimerization of complementary Fc polypeptides, see e.g. Ridgway etal, Protein Eng. 9:617-621 (1996); Merchant et al, Nat. Biotechnol.16(7): 677-81 (1998); Moore et al. (2011) MAbs, 3:546-57; VonKreudenstein et al. MAbs, (2013) 5:646-54; Gunasekaran et al. (2010) J.Biol. Chem., 285:19637-46; Leaver-Fay et al. (2016) Structure,24:641-51; Ha et al. (2016) Frontiers in Immunology, 7:1; Davis et al.(2010) Protein Eng Des Sel, 23:195-202; published international PCTAppl. No. WO 1998/050431, WO 2009/089004, WO2011143545 WO 2014/067011,WO 2012/058768, WO2018027025; published U.S. patent Appl. No.US20140363426, US20150307628, US20180016354, US20150239991; and U.S.patent Nos. U.S. Pat. Nos. 5,731,168, 7,183,076, 9,701,759, 9,605,084,and 9,650,446. Methods to promote heterodimerization of Fc chainsinclude mutagenesis of the Fc region, such as by including a set of“knob-into-hole” mutations or including mutations to effectelectrostatic steering of the Fc to favor attractive interactions amongdifferent polypeptide chains. For example, in some embodiments, the Fcpolypeptides of a heterodimer includes a mutation to alter chargepolarity across the Fc dimer interface such that coexpression ofelectrostatically matched Fc chains support favorable attractiveinteractions thereby promoting desired Fc heterodimer formation, whereasunfavorable repulsive charge interactions suppress unwanted Fc homodimerformation (Guneskaran et al. (2010) JBC, 285: 19637-19646). Whenco-expressed in a cell, association between the chains is possible butthe chains do not substantially self-associate due to charge repulsion.Other strategies for generating a heterodimeric Fc include mixing humanIgG and IgA CH3 domain segments to create a complementary CH3heterodimer, which is referred to as a SEED Fc.

Methods and variants for heterodimerization also include those describedin published international PCT App. WO2014/145806, including “knobs andholes” mutations (also called “skew” variants), mutations that relate to“electrostatic steering” or “charge pairs,” and pI variants.Heterodimeric variants also include any as described in U.S. publishedAppl. No. US2012/0149876 or US2018/011883.

In some embodiments, to promote heterodimerization both polypeptides ofthe Fc heterodimer contain paired or complementary amino acidmodifications. Exemplary paired amino acid modification of polypeptidesof an Fc fusion are set forth in Table 1.

TABLE 1 Paired amino acids of Heterodimeric Fc First Fc polypeptideSecond Fc Polypeptide T366W T366S/L368W/Y407V T366W/S354CT366S/L368A/Y407V/Y349C S364H/F405A Y349T/Y349F T350V/L351Y/F405A/Y407VT350V/T366L/K392L/T394W K360D/D399M/Y407A E345R/Q347R/T366V/K409VK409D/K392D D399K/E356K K360E/K409W Q347R/D399V/F405T L360E/K409W/Y349CQ347R/399V/F405T/S354C K370E/K409W E357N/D399V/F405T

In some embodiments, modifications include introduction of aprotuberance (knob) into a first Fc polypeptide and a cavity (hole) intoa second Fc polypeptide such that the protuberance is positionable inthe cavity to promote complexing of the first and second Fc-containingpolypeptides. Amino acids targeted for replacement and/or modificationto create protuberances or cavities in a polypeptide are typicallyinterface amino acids that interact or contact with one or more aminoacids in the interface of a second polypeptide.

In some embodiments, a first Fc polypeptide that is modified to containprotuberance (hole) amino acids include replacement of a native ororiginal amino acid with an amino acid that has at least one side chainwhich projects from the interface of the first Fc polypeptide and istherefore positionable in a compensatory cavity (hole) in an adjacentinterface of a second polypeptide. Most often, the replacement aminoacid is one which has a larger side chain volume than the original aminoacid residue. One of skill in the art knows how to determine and/orassess the properties of amino acid residues to identify those that areideal replacement amino acids to create a protuberance. In someembodiments, the replacement residues for the formation of aprotuberance are naturally occurring amino acid residues and include,for example, arginine (R), phenylalanine (F), tyrosine (Y), ortryptophan (W). In some examples, the original residue identified forreplacement is an amino acid residue that has a small side chain suchas, for example, alanine, asparagine, aspartic acid, glycine, serine,threonine, or valine.

In some embodiments, a second Fc polypeptide that is modified to containa cavity (hole) is one that includes replacement of a native or originalamino acid with an amino acid that has at least one side chain that isrecessed from the interface of the second polypeptide and thus is ableto accommodate a corresponding protuberance from the interface of afirst polypeptide. Most often, the replacement amino acid is one whichhas a smaller side chain volume than the original amino acid residue.One of skill in the art knows how to determine and/or assess theproperties of amino acid residues to identify those that are idealreplacement residues for the formation of a cavity. Generally, thereplacement residues for the formation of a cavity are naturallyoccurring amino acids and include, for example, alanine (A), serine (S),threonine (T) and valine (V). In some examples, the original amino acididentified for replacement is an amino acid that has a large side chainsuch as, for example, tyrosine, arginine, phenylalanine, or tryptophan.

The CH3 interface of human IgG1, for example, involves sixteen residueson each domain located on four anti-parallel β-strands which buries 1090Å2 from each surface (see e.g., Deisenhofer et al. (1981) Biochemistry,20:2361-2370; Miller et al., (1990) J Mol. Biol., 216, 965-973; Ridgwayet al., (1996) Prot. Engin., 9: 617-621; U.S. Pat. No. 5,731,168).Modifications of a CH3 domain to create protuberances or cavities aredescribed, for example, in U.S. Pat. No. 5,731,168; International PatentApplications WO98/50431 and WO 2005/063816; and Ridgway et al., (1996)Prot. Engin., 9: 617-621. In some examples, modifications of a CH3domain to create protuberances or cavities are typically targeted toresidues located on the two central anti-parallel β-strands. The aim isto minimize the risk that the protuberances which are created can beaccommodated by protruding into the surrounding solvent rather thanbeing accommodated by a compensatory cavity in the partner CH3 domain.

For example, in some embodiments the heterodimeric Fc includes apolypeptide having an amino acid modification within the CH3 domain atThr366, which when replaced with a more bulky amino acid, e.g., Try(T366W), is able to preferentially pair with a second CH3 domain havingamino acid modifications to less bulky amino acids at positions Thr366,Leu368, and Tyr407, e.g., Ser, Ala and Val, respectively(T366S/L368A/Y407V). Heterodimerization via CH3 modifications can befurther stabilized by the introduction of a disulfide bond, for exampleby changing Ser354 to Cys (S354C) and Tyr349 to Cys (Y349C) on oppositeCH3 domains (Reviewed in Carter, 2001 Journal of Immunological Methods,248: 7-15).

In particular embodiments, a multispecific polypeptide constructcontains a first and second Fc able to mediate Fc heterodimerizationcontains a first Fc polypeptide containing mutations T366W and S354C anda second Fc polypeptide containing mutations T366S, L368A, Y407V andY349C. In some embodiments, the first Fc polypeptide is selected from anFc polypeptide comprising the sequence set forth in SEQ ID NO: 201 or207 and the second Fc polypeptide is selected from an Fc polypeptidecomprising the sequence set forth in SEQ ID NO: 202, 205 or 209. In someembodiments, the first Fc polypeptide is or comprises the sequence ofamino acids set forth in any of SEQ ID NOS: 82, 86, 94 or 96 and thesecond Fc polypeptide is or comprises the sequence of amino acids setforth in any of SEQ ID NOS: 83, 87, 90, 92, 98 or 100.

In some embodiments, the Fc polypeptide exhibits features providingFc-mediated effector functions. In particular examples, the first Fcpolypeptide is or comprises the sequence set forth in SEQ ID NOs:201 anda second Fc polypeptide that is or comprises SEQ ID NO: 202 or 205. Insome embodiments, the first Fc polypeptide is or comprises the sequenceset forth in SEQ ID NO: 82 and the second Fc polypeptide is or comprisesthe sequence set forth in SEQ ID NO: 83 or 90. In some embodiments, thefirst Fc polypeptide is or comprises the sequence set forth in SEQ IDNO: 86 and the second Fc polypeptide is or comprises the sequence setforth in SEQ ID NO: 87 or 92. The first and second Fc polypeptide can beformatted on either polypeptide chain of the construct.

In some embodiments, one or both of the first and second Fc polypeptidescan further include one or more amino acid mutations to further reduceone or more Fc effector functions, such as reduced Fc receptor binding.Exemplary mutations to reduce Fc effector functions include any asdescribed. In some embodiments, the modification can be a deletion ofone or more positions Glu233 (E233), Leu234 (L234), or Leu235 (L235),such as a deletion of amino acids Glu233 (E233), Leu234 (L234), andLeu235 (L235). In some embodiments, the first Fc polypeptide is selectedfrom an Fc polypeptide comprising the sequence set forth in SEQ ID NO:203 or 208 and the second Fc polypeptide is selected from an Fcpolypeptide comprising the sequence set forth in SEQ ID NO: 204, 206 or210. In some embodiments, the first Fc polypeptide is or comprises thesequence of amino acids set forth in any of SEQ ID NOS: 84, 88, 95 or 97and the second Fc polypeptide is or comprises the sequence of aminoacids set forth in any of SEQ ID NOS: 85, 89, 91, 93, 99 or 101.

In particular examples, the first Fc polypeptide is or comprises thesequence set forth in SEQ ID NOs:203 and a second Fc polypeptide that isor comprises SEQ ID NO: 204 or 206. In some embodiments, the first Fcpolypeptide is or comprises the sequence set forth in SEQ ID NO: 84 andthe second Fc polypeptide is or comprises the sequence set forth in SEQID NO: 85 or 91. In some embodiments, the first Fc polypeptide is orcomprises the sequence set forth in SEQ ID NO: 88 and the second Fcpolypeptide is or comprises the sequence set forth in SEQ ID NO: 89 or93. The first and second Fc polypeptide can be formatted on eitherpolypeptide chain of the construct.

In some embodiments, the first Fc polypeptide or second Fc polypeptidefurther includes mutations M252Y and/or M428V. In particular examples,the first Fc polypeptide is or comprises the sequence set forth in SEQID NO:207 and the second Fc polypeptide is or comprises the sequence setforth in SEQ ID NO:209. In some embodiments, the first Fc polypeptide isor comprises the sequence set forth in SEQ ID NO:94 and the second Fcpolypeptide is or comprises the sequence set forth in SEQ ID NO: 98. Insome embodiments, the first Fc polypeptide is or comprises the sequenceset forth in SEQ ID NO:96 and the second Fc polypeptide is or comprisesthe sequence set forth in SEQ ID NO: 100. In other examples, the firstFc polypeptide is or comprises the sequence set forth in SEQ ID NO:208and the second Fc polypeptide is or comprises the sequence set forth inSEQ ID NO:210. In some embodiments, the first Fc polypeptide is orcomprises the sequence set forth in SEQ ID NO:95 and the second Fcpolypeptide is or comprises the sequence set forth in SEQ ID NO: 99. Insome embodiments, the first Fc polypeptide is or comprises the sequenceset forth in SEQ ID NO:97 and the second Fc polypeptide is or comprisesthe sequence set forth in SEQ ID NO: 101. The first and second Fcpolypeptide can be formatted on either polypeptide chain of theconstruct.

Additional examples of variants that can facilitate the promotion ofheterodimers are any combination or pair of steric variants (e.g. skewvariants) of a first Fc polypeptide and a second Fc polypeptide fromamong: S364K/E357Q and L368D/K370S; L368D/K370S and S364K; L368E/K370Sand S364K; T411T/E360E/Q362E and D401K; L368D/K370S and S364K/E357L,K370S and S364K/E357Q and T366S/L368A/Y407V and T366W or366S/L368A/Y407V/Y349C and T366W/S354C), where each pair representsmutations in the first Fc polypeptide and second Fc polypeptide. Inparticular embodiments, a provided construct contains a first and secondFc polypeptide containing the pair of mutations L368D/K370S and S364Kand E357Q.

An additional mechanism that can be used in the generation ofheterodimers is sometimes referred to as “electrostatic steering” asdescribed in Gunasekaran et al., J. Biol. Chem. 285(25):19637 (2010).This is sometimes referred to herein as “charge pairs”. In thisembodiment, electrostatics are used to skew the formation towardsheterodimerization. As those in the art will appreciate, these may alsohave an effect on pI, and thus on purification, and thus could in somecases also be considered pI variants. However, as these were generatedto force heterodimerization and were not used as purification tools,they are classified as “steric variants”. In one embodiments, a first Fcpolypeptide can contain mutations D221E/P228E/L368E and a second Fcpolypeptide can contain mutations D221R/P228R/K409R. In anotherembodiments, a first Fc polypeptide can contain mutationsC220E/P228E/368E and a second Fc polypeptide can contain mutationsC220R/E224R/P228R/K409R.

In some embodiments, heterodimerization can be facilitated by pIvariants. In some aspects, a pI variant can include those that increasethe pI of the protein (basic changes). In other aspects, the pI variantcan include those that decrease the pI of the protein (acidic changes).In some cases, all combinations of these variants can be done, includingcombinations in which one Fc polypeptide may be wild type, or a variantthat does not display a significantly different p1 from wild-type, andthe other Fc polypeptide can be either more basic or more acidic.Alternatively, each Fc polypeptide can be changed, one to more basic andone to more acidic. In some embodiments, at least one Fc polypeptide isa negative pI variant Fc containing mutations Q295E/N384D/Q418E/N421D.

In some embodiments, a combination of steric heterodimerization variants(e.g. knob and hole) and pI or charge pair variants can be used.

In particular embodiments, the provided constructs contains (a) a firstFc polypeptide comprising the skew variants S364K/E357Q; and b) a secondFc polypeptide containing skew variants L368D/K370S and the pI variantsN208D/Q295E/N384D/Q418E/N421D. In some embodiments, one or both of thefirst and second polypeptide can contain further mutations to reduce Fceffector activity, such as the exemplary mutationsE233P/L234V/L235A/G236del/S267K. An example of such a first Fcpolypeptide and a second Fc polypeptide able to mediate Fcheterodimeriztion comprise the sequences set forth in SEQ ID NOs:194 and195. The first and second Fc polypeptide can be formatted on eitherpolypeptide chain of the construct.

The resulting multispecific polypeptide constructs can be purified byany suitable method such as, for example, by affinity chromatographyover Protein A or Protein G columns. Where two nucleic acid moleculesencoding different polypeptides are transformed into cells, formation ofhomo- and heterodimers will occur. Conditions for expression can beadjusted so that heterodimer formation is favored over homodimerformation.

Techniques for recovery of heterodimers from homodimers based on adifferential affinity of the heterodimers for an affinity reagent areknown. In some aspects, such techniques include designing a heterodimerso that one of the Fc polypeptide chains does not bind to the affinityreagent protein A. In some cases, one of the polypeptide chain cancontain one or more amino acid substitution to abrogate or reduceaffinity for the protein A reagent in one of the polypeptides of the Fcheterodimer, see e.g. WO2017134440, WO2010151792, Jendeberg et al.(Jendeberg et al., (1997) J. Immunol. Methods, 201(1): 25-34. In some ofthese embodiments, the Fc region may be modified at the protein-Abinding site on one member of the heterodimer so as to prevent protein-Abinding and thereby enable more efficient purification of theheterodimeric fusion protein. An exemplary modification within thisbinding site is Ile253, for example Ile253Arg (I253R). In someembodiments, the modification may be H435R or H435R/Y436F. In someembodiments, an Fc polypeptide of an Fc heterodimer can contain amodification so that it is capable of binding protein A but not proteinG (pA+/pG−). Exemplary pA+/pG− amino acid modifications include an Fccontaining serine at position 428, serine at position 434 and optionallyhistidine at position 436, with reference to human IgG1 or comprisingthese residues at the corresponding positions in human IgG 2, 3, or 4.In some aspects, such amino acid modifications in one IgG Fc polypeptideat positions 428, 434 and optionally 436 reduces or prevents the bindingof protein G, enhancing the purification of the protein.

In some embodiments, any of such modifications to confer differentialaffinity to an affinity reagent can be combined with any one or moreother amino acid modifications described above. For example, the I253Rmodification maybe combined with either the T366S/L368A/Y407Vmodifications or with the T366W modifications. The T366S/L368A/Y407Vmodified Fc is capable of forming homodimers as there is no stericocclusion of the dimerization interface as there is in the case of theT336W modified Fc. Therefore, in some embodiments, the I253Rmodification is combined with the T366S/L368A/Y407V modified Fc todisallow purification any homodimeric Fc that may have formed. Similarmodifications can be employed by combining T366S/L368A/Y407V and H453R.

In some embodiments, the Fc regions of the heterodimeric moleculeadditionally can contain one or more other Fc mutation, such as anydescribed above. In some embodiments, the heterodimer molecule containsan Fc region with a mutation that reduces effector function.

In some embodiments, one Fc polypeptide of a heterodimeric Fc comprisesthe sequence of amino acids set forth in any of SEQ ID NOS:201 (e.g. SEQID NO:82), 86, 207 (e.g. SEQ ID NO:94), or 96, and the other Fcpolypeptide of the heterodimeric Fc contains the sequence of amino acidsset forth in any of SEQ ID NOS:201 (e.g. SEQ ID NO:83), 87, 205 (e.g.SEQ ID NO:90), 92, 209 (e.g. SEQ ID NO:98), or 100. In some embodiments,one Fc polypeptide of a heterodimeric Fc comprises the sequence of aminoacids set forth in any of SEQ ID NOS: 203 (e.g. SEQ ID NO:84), 88, 208(e.g. SEQ ID NO:95), or 97 and the other Fc polypeptide of theheterodimeric Fc comprises the sequence of amino acids set forth in anyof SEQ ID NOS: 204 (e.g. SEQ ID NO:85), 89, 206 (e.g. SEQ ID NO:91), 93,210 (e.g. SEQ ID NO:99), or 101.

In some embodiments, the human IgG Fc region is modified to preventdimerization. In these embodiments, the fusion proteins of the presentdisclosure are monomeric. For example modification at residue Thr366 toa charged residue, e.g. Thr366Lys, Thr366Arg, Thr366Asp, or Thr366Glu(T366K, T366R, T366D, or T366E, respectively), prevents CH3-CH3dimerization.

In some embodiments, the Fc region of the fusion protein is altered atone or more of the following positions to reduce Fc receptor binding:Leu 234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Ser298(S298), Asn297 (N297), Asn325 (N325) orAla327 (A327). For example, Leu234Ala (L234A), Leu235Ala (L235A), Asp265Asn (D265N), Asp270Asn (D270N),Ser298Asn (S298N), Asn297Ala (N297A), Asn325Glu (N325E) orAla327Ser(A327S). In preferred embodiments, modifications within the Fc regionreduce binding to Fc-receptor-gamma receptors while have minimal impacton binding to the neonatal Fc receptor (FcRn).

In some embodiments, the fusion protein contains a polypeptide derivedfrom an immunoglobulin hinge region. The hinge region can be selectedfrom any of the human IgG subclasses. For example, the fusion proteinmay contain a modified IgG1 hinge having the sequence of EPKSSDKTHTCPPC(SEQ ID NO: 7), where in the Cys220 that forms a disulfide with theC-terminal cysteine of the light chain is mutated to serine, e.g.,Cys220Ser (C220S). In other embodiments, the fusion protein contains atruncated hinge having a sequence DKTHTCPPC (SEQ ID NO: 8).

In some embodiments, the fusion protein has a modified hinge from IgG4,which is modified to prevent or reduce strand exchange, e.g., Ser228Pro(S228P), having the sequence ESKYGPPCPPC (SEQ ID NO: 9). In someembodiments, the fusion protein contains linker polypeptides. In otherembodiments, the fusion protein contains linker and hinge polypeptides.

3. Linkers

The provided multispecific polypeptide constructs contain a linker thatjoins or couples the first component containing the immunoglobulin Fcregion and the second component containing the CD3 binding region. Insome embodiments, the linker is a non-cleavable linker. In someembodiments, the linker does not contain a substrate recognition sitethat is specifically recognized for cleavage by the protease. Thus,linkers in the provided multispecific polypeptide constructs do notinclude an amino acid sequence that can serve as a substrate for aprotease, such as an extracellular protease. For example, thenon-cleavable linker does not include a cleavage sequence containing atleast one peptide bond which lies within a cleavable peptide sequence ofa protease.

In some embodiments, the linker is positioned at the end of theC-terminal region of the Fc region, such that the Fc region isN-terminal to the CD3 binding region. Because the provided multispecificpolypeptide constructs are multimers, such as dimers, the providedconstructs include a linker joining the first Fc polypeptide and a firstdomain (e.g. VH) of the CD3 binding region of the first polypeptide andthe second Fc polypeptide and second domain (e.g. VL) of the CD3 bindingregion of the second polypeptide. Typically, the linkers present in thefirst and second polypeptides of the multispecific polypeptide constructare the same. Thus, in some embodiments, each domain of the CD3 bindingdomain is linked via a linker, such as the same linker, to oppositepolypeptides of the Fc, such as heterodimeric Fc.

Various polypeptide linkers for use in fusion proteins are known (seee.g. Chen et al. (2013) Adv. Drug. Deliv. 65:1357-1369; andInternational PCT publication No. WO 2014/099997, WO2000/24884; U.S.Pat. Nos. 5,258,498; 5,525,491; 5,525,491, 6,132,992).

In some embodiments, the linker is chosen so that, when the CD3 bindingregion is joined to the Fc region of the multispecific polypeptideconjugate, the CD3 binding region is constrained and not able to, or notsubstantially able to, bind or engage CD3 on the surface of a cell, e.g.T cell, upon contact of the multispecific polypeptide construct with thecell. Various assays can be employed to assess binding or engagement ofCD3 by the multispecific polypeptide construct, including assays toassess T cell binding, NFAT activation using a reporter system,cytolytic T cell activity, cytokine production and/or expression of Tcell activation markers. Exemplary assays are shown in the providedExamples. Typically, the linker also is one that ensures correct foldingof the polypeptide construct, does not exhibit a charge that would beinconsistent with the activity or function of the linked polypeptides orform bonds or other interactions with amino acid residues in one or moreof the domains that would impede or alter activity of the linkedpolypeptides. In some embodiment, the linker is a polypeptide linker.The polypeptide linker can be a flexible linker or a rigid linker or acombination of both.

In some aspects, the linker is a short, medium or long linker. In someembodiments, the linker is up to 40 amino acids in length. In someembodiments, the linker is up to 25 amino acids in length. In someembodiments, the linker is at least or is at least about 2 amino acidsin length. In some aspects, a suitable length is, e.g., a length of atleast one and typically fewer than about 40 amino acid residues, such as2-25 amino acid residues, 5-20 amino acid residues, 5-15 amino acidresidues, 8-12 amino acid. In some embodiments, the linker is from orfrom about 2 to 24 amino acids, 2 to 20 amino acids, 2 to 18 aminoacids, 2 to 14 amino acids, 2 to 12 amino acids, 2 to 10 amino acids, 2to 8 amino acids, 2 to 6 amino acids, 6 to 24 amino acids, 6 to 20 aminoacids, 6 to 18 amino acids, 6 to 14 amino acids, 6 to 12 amino acids, 6to 10 amino acids, 6 to 8 amino acids, 8 to 24 amino acids, 8 to 20amino acids, 8 to 18 amino acids, 8 to 14 amino acids, 8 to 12 aminoacids, 8 to 10 amino acids, 10 to 24 amino acids, 10 to 20 amino acids,10 to 18 amino acids, 10 to 14 amino acids, 10 to 12 amino acids, 12 to24 amino acids, 12 to 20 amino acids, 12 to 18 amino acids, 12 to 14amino acids, 14 to 24 amino acids, 14 to 20 amino acids, 14 to 18 aminoacids, 18 to 24 amino acids, 18 to 20 amino acids or 20 to 24 aminoacids. In some embodiments, the linker is 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length.

In certain aspects, the longer the linker length, the greater the CD3binding when the multispecific polypeptide conjugate is bounds to itsantigen, e.g. TAA. Thus, in some aspects, the linker is greater than 12amino acids in length, such as greater than 13, 14, 15, 16, 17 or 18amino acids in length. In some embodiments, the linker is 12 to 40 aminoacids in length, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18acids, 12 to 15 amino acids, 15 to 40 amino acids, 15 to 30 amino acids,15 to 24 amino acids, 15 to 18 amino acids, 18 to 40 amino acids, 18 to30 amino acids, 18 to 24 amino acids, 24 to 40 amino acids, 24 to 30amino acids or 30 to 40 amino acids.

The linkers can be naturally-occurring, synthetic or a combination ofboth. Particularly suitable linker polypeptides predominantly includeamino acid residues selected from Glycine (Gly), Serine (Ser), Alanine(Ala), and Threonine (Thr). For example, the linker may contain at least75% (calculated on the basis of the total number of residues present inthe peptide linker), such as at least 80%, at least 85%, or at least 90%of amino acid residues selected from Gly, Ser, Ala, and Thr. The linkermay also consist of Gly, Ser, Ala and/or Thr residues only. In someembodiments, the linker contains 1-25 glycine residues, 5-20 glycineresidues, 5-15 glycine residues, or 8-12 glycine residues. In someaspects, suitable peptide linkers typically contain at least 50% glycineresidues, such as at least 75% glycine residues. In some embodiments, apeptide linker comprises glycine residues only. In some embodiments, apeptide linker comprises glycine and serine residues only.

In some embodiments, these linkers are composed predominately of theamino acids Glycine and Serine, denoted as GS-linkers herein. In someembodiments, the linker contains (GGS)n, wherein n is 1 to 10, such as 1to 5, for example 1 to 3, such as GGS(GGS)n (SEQ ID NO:171), wherein nis 0 to 10. In particular embodiments, the linker contains the sequence(GGGGS)n (SEQ ID NO: 173), wherein n is 1 to 10 or n is 1 to 5, such as1 to 3. In further embodiments, the linker contains (GGGGGS)n (SEQ IDNO:172), wherein n is 1 to 4, such as 1 to 3. The linker can includecombinations of any of the above, such as repeats of 2, 3, 4, or 5 GS,GGS, GGGGS, and/or GGGGGS linkers may be combined. In some embodiments,such a linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18 or 19 amino acids in length.

In some embodiments, the linker is (in one-letter amino acid code): GGS,GGGGS (SEQ ID NO: 149), or GGGGGS (SEQ ID NO: 135). In some embodiments,the GS-linker comprises an amino acid sequence of GGSGGS, i.e., (GGS)₂(SEQ ID NO: 10); GGSGGSGGS, i.e., (GGS)₃ (SEQ ID NO: 11); GGSGGSGGSGGS,i.e., (GGS)₄ (SEQ ID NO: 12); GGSGGSGGSGGSGGS, i.e., (GGS)₅ (SEQ ID NO:13); GGGGGSGGGGGSGGGGGS, i.e., (G5S)₃ (SEQ ID NO: 119),GGSGGGGSGGGGSGGGGS (SEQ ID NO: 147) and GGGGSGGGGSGGGGS (SEQ ID NO:170).In some embodiments, the linker is GGGG (SEQ ID NO:103). In someembodiments, the linker is GGGGG (SEQ ID NO:192). In some of any of theabove examples, serine can be replaced with alanine (e.g., (Gly4Ala) or(Gly3Ala)).

In some embodiments, the linker includes a peptide linker having theamino acid sequence Gly_(x)Xaa-Gly_(y)-Xaa-Gly_(z) (SEQ ID NO:174),wherein each Xaa is independently selected from Alanine (Ala), Valine(Val), Leucine (Leu), Isoleucine (Ile), Methionine (Met), Phenylalanine(Phe), Tryptophan (Trp), Proline (Pro), Glycine (Gly), Serine (Ser),Threonine (Thr), Cysteine (Cys), Tyrosine (Tyr), Asparagine (Asn),Glutamine (Gln), Lysine (Lys), Arginine (Arg), Histidine (His),Aspartate (Asp), and Glutamate (Glu), and wherein x, y, and z are eachintegers in the range from 1-5. In some embodiments, each Xaa isindependently selected from the group consisting of Ser, Ala, and Thr.In a specific variation, each of x, y, and z is equal to 3 (therebyyielding a peptide linker having the amino acid sequenceGly-Gly-Gly-Xaa-Gly-Gly-Gly-Xaa-Gly-Gly-Gly (SEQ ID NO:175), whereineach Xaa is selected as above.

In some embodiments, the linker is serine-rich linkers based on therepetition of a (SSSSG)n (SEQ ID NO:185) motif where n is at least 1,though n can be 2, 3, 4, 5, 6, 7, 8 and 9.

In some cases, it may be desirable to provide some rigidity into thepeptide linker. This may be accomplished by including proline residuesin the amino acid sequence of the peptide linker. Thus, in someembodiments, a linker comprises at least one proline residue in theamino acid sequence of the peptide linker. For example, a peptide linkercan have an amino acid sequence wherein at least 25% (e.g., at least 50%or at least 75%) of the amino acid residues are proline residues. In oneparticular embodiment, the peptide linker comprises proline residuesonly.

In some aspects, a peptide linker comprises at least one cysteineresidue, such as one cysteine residue. For example, in some embodiments,a linker comprises at least one cysteine residue and amino acid residuesselected from the group consisting of Gly, Ser, Ala, and Thr. In somesuch embodiments, a linker comprises glycine residues and cysteineresidues, such as glycine residues and cysteine residues only.Typically, only one cysteine residue will be included per peptidelinker. One example of a specific linker comprising a cysteine residueincludes a peptide linker having the amino acid sequenceGly_(m)-Cys-Gly_(n), wherein n and m are each integers from 1-12, e.g.,from 3-9, from 4-8, or from 4-7. In a specific variation, such a peptidelinker has the amino acid sequence GGGGG-C-GGGGG (SEQ ID NO:177).

In some embodiments, the linker of the fusion protein is a structured orconstrained linker. In particular embodiments, the structured linkercontains the sequence (AP)n or (EAAAK)n (SEQ ID NO:178), wherein n is 2to 20, preferably 4 to 10, including but not limited to, AS-(AP)n-GT(SEQ ID NO:179) or AS-(EAAAK)n-GT (SEQ ID NO:180), wherein n is 2 to 20,such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In otherembodiments, the linker comprises the sequences (GGGGA)n (SEQ IDNO:181), (PGGGS)n (SEQ ID NO:182), (AGGGS)n (SEQ ID NO:183) orGGS-(EGKSSGSGSESKST)n-GGS (SEQ ID NO:184, wherein n is 2 to 20. In someembodiments, the linker is SSSASASSA (SEQ ID NO:186), GSPGSPG (SEQ IDNO:187), or ATTTGSSPGPT (SEQ ID NO:176). In some embodiments, suchlinkers, by virtue of their structure, may be more resistant toproteolytic degradation, thereby offering an advantage when injected invivo.

In some embodiments, the linker is not a cleavable linker (usedinterchangeably with non-cleavable linker). In some embodiments, thelinker is not cleavable by a protease. In some embodiments, a linkerthat is not a cleavable linker or that is not cleavable by a protease isone that is generally stable for in vivo delivery or recombinantproduction. In some aspects, a linker that is not cleavable by aprotease includes those that do not contain at least one peptide bondwhich preferably lies within a cleavable peptide sequence or recognitionsite of a protease. In particular embodiments, a non-cleavable linker isnot a target substrate for a protease, such that it is notpreferentially or specifically cleaved by a protease compared to alinker that contains a substrate recognition site for the same protease.

In some embodiments, the linker does not contains a substraterecognition site or cleavage site for a particular protease, which isthe sequence recognized by the active site of a protease that is cleavedby a protease. Typically, for example, for a serine protease, a cleavagesequence is made up of the P1-P4 and P1′-P4′ amino acids in a substrate,where cleavage occurs after the P1 position. Typically, a cleavagesequence for a serine protease is six residues in length to match theextended substrate specificity of many proteases, but can be longer orshorter depending upon the protease. Typically, the linker does notinclude a P1-P1′ scissile bond sequence that is recognized by aprotease.

In some aspects, a non-cleavable linker or a linker that does notcontain a substrate recognition site that is specifically recognized forcleavage by a protease is one whose cleavage by a protease issubstantially less than cleavage of a target substrate of the protease.Typically, a protease exhibits specificity or preference for cleavage ofa particular target substrate compared to another non-target substrate.Such a degree of specificity can be determined based on the rateconstant of cleavage of a sequence, e.g. linker sequence, which is ameasure of preference of a protease for its substrate and the efficiencyof the enzyme. Any method to determine the rate of increase of cleavageover time in the presence of various concentrations of substrate can beused to calculate the specificity constant. For example, a substrate islinked to a fluorogenic moiety, which is released upon cleavage by aprotease. By determining the rate of cleavage at different proteaseconcentrations the specificity constant for cleavage (k_(cat)/K_(m)) canbe determined for a particular protease towards a particular linker. Insome embodiments, a non-cleavable linker, or a linker that does notcontain a substrate recognition site that is specifically recognized forcleavage by a protease, is a linker that, if cleaved at all, is cleavedby a protease at a rate of less than 1×10⁴ M⁻¹S⁻, or less than 5×10³M⁻¹S, less than 1×10³ M⁻¹S, or less than 1×10² M⁻¹S or less.

In some embodiments, the linkers in the multispecific constructsprovided herein do not contain a substrate recognition site for aprotease that include, for example, matrix metalloproteases (MMP),cysteine proteases, serine proteases and plasmin activators. Inparticular embodiments, the linker does not contain a substraterecognition site for a protease that is a protease that is produced by atumor, an activated immune effector cell (e.g. a T cell or a NK cell),or a cell in a tumor microenvironment.

In some embodiments, the linker does not contain a substrate recognitionsite that is specifically recognized by one or more of the followingenzymes or proteases: ADAMS, ADAMTS, e.g. ADAMS; ADAMS; ADAM10; ADAM12;ADAM15; ADAM17/TACE; ADAMDEC1; ADAMTS1; ADAMTS4; ADAMTSS; aspartateproteases, e.g., BACE or Renin; aspartic cathepsins, e.g., Cathepsin Dor Cathepsin E; Caspases, e.g., Caspase 1, Caspase 2, Caspase 3, Caspase4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, orCaspase 14; cysteine cathepsins, e.g., Cathepsin B, Cathepsin C,Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V/L2, Cathepsin X/Z/P;Cysteine proteinases, e.g., Cruzipain; Legumain; Otubain-2; KLKs, e.g.,KLK4, KLKS, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, or KLK14; Metalloproteinases, e.g., Meprin; Neprilysin; PSMA; BMP-1; MMPs, e.g., MMP1,MMP2, MMP3, MMP1, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15,MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, or MMP27, serineproteases, e.g., activated protein C, Cathepsin A, Cathepsin G, Chymase,coagulation factor proteases (e.g., FVIIa, FIXa, FXa, FXIa, FXIIa),Elastase, Granzyme B, Guanidinobenzoatase, HtrA1, Human NeutrophilElastase, Lactoferrin, Marapsin, NS3/4A, PACE4, Plasmin, PSA, tPA,Thrombin, Tryptase, uPA; Type II Transmembrane Serine Proteases (TTSPs),e.g., DESC1, DPP-4, FAP, Hepsin, Matriptase-2, Matriptase, TMPRSS2,TMPRSS3, or TMPRSS4; and any combination thereof. In some embodiments,the linker does not contain a substrate recognition site that isspecifically recognized by granzyme B, a matriptase or an MMP, such asMMP-2.

In some embodiments, the linker does not comprise an amino acid that isa substrate for Granzyme B. In some embodiments, the linker does notcontain an amino acid sequence having the general formula P4 P3 P2P1↓P1′ (SEQ ID NO: 150), wherein P4 is amino acid I, L, Y, M, F, V, orA; P3 is amino acid A, G, S, V, E, D, Q, N, or Y; P2 is amino acid H, P,A, V, G, S, or T; P1 is amino acid D or E; and P1′ is amino acid I, L,Y, M, F, V, T, S, G or A. In some embodiments, the linker does notcontain an amino acid sequence having the general formula P4 P3 P2P1↓P1′ (SEQ ID NO: 151), wherein P4 is amino acid I or L; P3 is aminoacid E; P2 is amino acid P or A; P1 is amino acid D; and P1′ is aminoacid I, V, T, S, or G.

In some embodiments, the linker does not contain the amino acid sequenceLEAD (SEQ ID NO: 22), LEPD(SEQ ID NO: 142), or LEAE (SEQ ID NO:143). Insome embodiments, the linker does not contain the amino acid sequenceIEPDI (SEQ ID NO:136), LEADT (SEQ ID NO:137), IEPDG (SEQ ID NO:138),IEPDV (SEQ ID NO:139), IEPDS (SEQ ID NO:140), IEPDT (SEQ ID NO:141),IEPDP (SEQ ID NO:144), LEPDG (SEQ ID NO:152) or LEADG (SEQ ID NO:153).

In some embodiments, the linker does not comprise an amino acid that isa substrate for matriptase. In some embodiments, the linker does notcomprises the sequence P1QAR↓(A/V) (SEQ ID NO: 154), wherein P1 is anyamino acid. In some embodiments, the linker does not comprises thesequence RQAR(A/V) (SEQ ID NO: 155). In some embodiments, the linkerdoes not comprise the amino acid sequence RQAR (SEQ ID NO: 23). In someembodiments, the linker does not comprise the amino acid sequence RQARV(SEQ ID NO: 156)

In some embodiments, the linker does not comprise an amino acid that isa substrate for one or more matrix metalloproteases (MMPs). In someembodiments, the MMP is MMP-2. In some embodiments, the linker does notcontain a sequence having the general formula P3 P2 P1↓P1′ (SEQ ID NO:157), wherein P3 is P, V or A; P2 is Q or D; P1 is A or N; and P1′ is L,I or M. In some embodiments, the linker does not contain the generalformula P3 P2 P1↓P1′ (SEQ ID NO: 158), wherein P3 is P; P2 is Q or D; P1is A or N; and P1′ is L or I. In some embodiments, the linker does notcomprise the amino acid sequence PAGL (SEQ ID NO: 24).

In some embodiments, the linker is not a linker comprising the aminoacid sequence set forth as TGLEADGSPAGLGRQARVG (SEQ ID NO: 25);TGLEADGSRQARVGPAGLG (SEQ ID NO: 26); TGSPAGLEADGSRQARVGS (SEQ ID NO:27); TGPAGLGLEADGSRQARVG (SEQ ID NO: 28); TGRQARVGLEADGSPAGLG (SEQ IDNO: 29); TGSRQARVGPAGLEADGS (SEQ ID NO: 30); and TGPAGLGSRQARVGLEADGS(SEQ ID NO: 31); GPAGLGLEPDGSRQARVG (SEQ ID NO: 104); GGSGGGGIEPDIGGSGGS(SEQ ID NO: 105); GGSGGGGLEADTGGSGGS (SEQ ID NO: 106); GSIEPDIGS (SEQ IDNO: 107); GSLEADTGS (SEQ ID NO: 108); GGSGGGGIEPDGGGSGGS (SEQ ID NO:109); GGSGGGGIEPDVGGSGGS (SEQ ID NO: 110); GGSGGGGIEPDSGGSGGS (SEQ IDNO: 111); GGSGGGGIEPDTGGSGGS (SEQ ID NO: 112); GGGSLEPDGSGS (SEQ ID NO:113); and GPAGLGLEADGSRQARVG (SEQ ID NO: 114), GGEGGGGSGGSGGGS (SEQ IDNO: 115); GSSAGSEAGGSGQAGVGS (SEQ ID NO: 116); GGSGGGGLEAEGSGGGGS (SEQID NO: 117); GGSGGGGIEPDPGGSGGS(SEQ ID NO: 118); TGGSGGGGIEPDIGGSGGS(SEQ ID NO: 148).

4. Antigen Binding Domains:

The multispecific polypeptide constructs of the present disclosureinclude at least one antigen binding domain, such as at least a firstantigen binding domain and a second antigen binding domain. In someaspects, the antigen binding domain, or independently each of theantigen binding domains, is selected from an antibody or antigen bindingfragment, a natural cognate binding partner, an Anticalin (engineeredlipocalin), a Darpin, a Fynomer, a Centyrin (engineered fibroneticin IIIdomain), a cystine-knot domain, an Affilin, an Affibody, or anengineered CH3 domain. In some embodiments, the natural cognate bindingpartner comprises an extracellular domain or binding fragment thereof ofthe native cognate binding partner of the TAA, or a variant thereof thatexhibits binding activity to the TAA.

In some embodiments, a TAA is a counter-structure that is presentprimarily on tumor cells of a mammalian subject but generally not foundon normal cells of the mammalian subject. A tumor specific antigen neednot be exclusive to tumor cells but the percentage of cells of aparticular mammal that have the tumor associated antigen is sufficientlyhigh or the levels of the tumor associated antigen on the surface of thetumor are sufficiently high such that it can be targeted by anti-tumortherapeutics, such as multispecific polypeptide constructs as provided,and provide prevention or treatment of the mammal from the effects ofthe tumor. In some embodiments, in a random statistical sample of cellsfrom a mammal with a tumor, at least 50% of the cells displaying a TAAare cancerous. In other embodiments, at least 60%, 70%, 80%, 85%, 90%,95%, or 99% of the cells displaying a TAA are cancerous.

In some embodiments, the antigen binding domain, or independently eachof the antigen binding domains, such as the first antigen-binding domainand the second antigen binding domains, includes one or more copies ofan antibody or an antigen-binding fragment thereof. In some embodiments,the antigen binding domain, or independently each of the antigen bindingdomains, includes one or more copies of an antibody or anantigen-binding fragment thereof selected from the group consisting of aFab fragment, a F(ab′)₂ fragment, an Fv fragment, a scFv, a scAb, a dAb,a single domain heavy chain antibody, and a single domain light chainantibody. In some embodiments, the antigen binding domain, orindependently each of the antigen binding domains, such as the firstantigen-binding domain and the second antigen binding domains, is asingle chain antibody. In some examples, the single chain is an scFv, ascAb, a single domain heavy chain antibody, or a single domain lightchain antibody. In some embodiments, each of the first antigen-bindingdomain and the second antigen binding domains includes one or moresingle domain antibody (sdAb) fragments, for example V_(H)H, V_(NAR),engineered V_(H) or V_(K) domains. V_(H)Hs can be generated from naturalcamelid heavy chain only antibodies, genetically modified rodents thatproduce heavy chain only antibodies, or naïve/synthetic camelid orhumanized camelid single domain antibody libraries. V_(NAR)s can begenerated from cartilaginous fish heavy chain only antibodies. Variousmethods have been implemented to generate monomeric sdAbs fromconventionally heterodimeric V_(H) and V_(K) domains, includinginterface engineering and selection of specific germline families.

In some embodiments, the antigen binding domain or independently each ofthe antigen binding domains, such as the first antigen-binding domainand/or the second antigen binding domains, of the multispecificpolypeptide constructs contains at least one sdAb or an scFv that bindsa TAA. In some embodiments, the at least one scFv or sdAb that binds aTAA is positioned amino-terminally relative to the Fc region and/orcarboxy-terminally relative to the CD3 binding region of themultispecific polypeptide construct. In some embodiments, themultispecific polypeptide construct contains only one scFv or sdAb thatbinds to a TAA, which can be positioned either amino-terminally relativeto the Fc region and/or carboxy-terminally relative to the CD3 bindingregion. In some embodiments, the multispecific polypeptide constructcontains two scFvs or sdAbs that bind to a TAA, positionedamino-terminally relative to the Fc region and/or carboxy-terminallyrelative to the CD3 binding region. In some embodiments, themultispecific polypeptide construct contains three scFv or sdAb, inwhich two are positioned amino-terminally relative to the Fc region orcarboxy-terminally relative to the CD3 binding region, and the third ispositioned at the other end of the multispecific polypeptide construct.

In some embodiments, the multispecific polypeptide construct is formedfrom or includes two polypeptides, including a first polypeptidecomprising a first Fc polypeptide of a heterodimeric Fc region, alinker, a VH domain of an anti-CD3 antibody or antigen binding fragment(e.g. Fv), and an scFv or sdAb that binds to a tumor-associated antigen;and a second polypeptide comprising a second Fc polypeptide of theheterodimeric Fc region, the linker, a VL domain of the anti-CD3antibody or antigen binding fragment (e.g. Fv) and, optionally, the sameor different scFv or sdAb that binds to a tumor-associated antigen. ThescFv or sdAb that binds to a TAA can be positioned amino terminallyrelative to an Fc polypeptide of the heterodimeric Fc and/orcarboxy-terminally relative to a VH or VL chain of the CD3 bindingregion. In some embodiments, the antigen binding domain, orindependently each of the antigen binding domains, of the multispecificpolypeptide constructs contains VH and VL sequences assembled as FABs orscFvs. In some embodiments, the antigen binding domain, or independentlyeach of the antigen binding domains, of the multispecific polypeptideconstructs contains binding domains as single domain antibodies (sdAbs).

In some embodiments, the antigen binding domain or independently each ofthe antigen binding domains, such as the first antigen-binding domainand the second antigen binding domains, contains more than one chain. Insome embodiments, the antigen binding domain or independently each ofthe antigen binding domains, such as the first antigen-binding domainand/or the second antigen binding domains, of the multispecificpolypeptide constructs contains VH and VL sequences assembled as FABs.

In some embodiments, the antigen binding domain or independently each ofthe antigen binding domains, such as the first antigen-binding domainand/or the second antigen binding domains, of the multispecificpolypeptide constructs contains a VH-CH1 (Fd) and a VL-CL of a Fabantibody that binds a TAA. In some embodiments, the Fab antibodycontaining a VH-CH1 (Fd) and a VL-CL is positioned amino-terminallyrelative to the Fc region and/or carboxy-terminally relative to the CD3binding region of the multispecific polypeptide construct. In someembodiments, the multispecific polypeptide construct contains only oneFab antibody, containing a VH-CH1 (Fd) and VL-CL, that binds to a TAA,which can be positioned either amino-terminally relative to the Fcregion and/or carboxy-terminally relative to the CD3 binding region. Insome embodiments, the multispecific polypeptide construct contains twoFab antibody fragments, each containing a VH-CH1 (Fd) and VL-CL, thatbinds to a TAA, in which one is positioned amino-terminally relative tothe Fc region and the other is positioned carboxy-terminally relative tothe CD3 binding region.

In some embodiments, the multispecific polypeptide construct is formedfrom or includes three or more polypeptides, including a firstpolypeptide comprising a first Fc polypeptide of a heterodimeric Fcregion, a linker and a VH-CH1 (Fd) or VL-CL of a Fab antibody fragmentthat binds to a tumor-associated antigen; a second polypeptidecomprising a second Fc polypeptide of the heterodimeric Fc region, thelinker and, optionally, the same VH-CH1 (Fd) or VL-CL of the Fabantibody fragment that binds to a tumor-associated antigen, and a thirdpolypeptide comprising the other of the VH-CH1 (Fd) or VL-CL of the Fabantibody fragment that binds to the TAA.

In some embodiments, the antigen binding domain, or independently eachof the antigen binding domains, is or includes an extracellular domainor binding fragment thereof of the native cognate binding partner of theTAA, or a variant thereof that exhibits binding activity to the TAA.

In some embodiments, each of the antigen binding domains, such as eachof the first antigen-binding domain and the second antigen bindingdomains, bind the same antigen. In some embodiments, each of the firstantigen-binding domain and the second antigen binding domains bind adifferent antigen. In some embodiments, each of the antigen bindingdomains, such as each of the first antigen-binding domain and the secondantigen binding domains, bind the same tumor associated antigen (TAA).In some embodiments, each of the antigen binding domains, such as eachof the first antigen-binding domain and the second antigen bindingdomains, bind a different TAA. In some embodiments, each of the antigenbinding domains, such as each of the first antigen-binding domain andthe second antigen binding domains, bind a different epitope on the sameTAA. In some embodiments, each of the antigen binding domains, such aseach of the first antigen-binding domain and the second antigen bindingdomains, bind the same epitope on the same TAA.

In some embodiments, the antigen binding domains results in monovalent,bivalent, trivalent, or tetravalent binding to the TAA. In someembodiments, bivalent binding to the TAA comprises two antigen bindingdomains that bind the same epitope of the same antigen (e.g.mono-epitopic). In some embodiments, bivalent binding to the TAAcomprises two antigen binding domains that bind different epitopes ofthe same antigen (e.g. bi-epitopic). In some embodiments, monovalentbinding to the TAA comprises one antigen binding domain that binds oneepitope of the antigen (e.g. mono-epitopic).

In some embodiments, the TAA is selected from the group consisting of1-92-LFA-3, 5T4, Alpha-4 integrin, Alpha-V integrin, alpha4beta1integrin, alpha4beta7 integrin, AGR2, Anti-Lewis-Y, Apelin J receptor,APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9,(Lewis a), Carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20,CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44,CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81,CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138,CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), CLAUDIN-3,CLAUDIN-4, cMet, Collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10,CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1,EDA, EDB, EGFR, EGFRviii, Endothelin B receptor (ETBR), ENPP3, EpCAM,EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2,FGFR3, FGFR4, FLT-3, Folate receptor alpha (FRa), GAL3ST1, G-CSF,G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GP IIb/IIIa receptors,Gp130, GPIIB/IIIA, GPNMB, GRP78, HER2/neu, HER3, HER4, HGF, hGH, HVEM,Hyaluronidase, ICOS, IFNalpha, IFNbeta, IFNgamma, IgE, IgE Receptor(FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R,IL-12Rbeta1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R,IL27/IL27R (wsx1), IL29, IL-31R, IL31/IL31R, IL2R, IL4, IL4R, IL6, IL6R,Insulin Receptor, Jagged Ligands, Jagged 1, Jagged 2, KISS1-R, LAG-3,LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, Mesothelin,MRP4, MUC1, Mucin-16 (MUC16, CA-125), Na/K ATPase, NGF, Nicastrin, NotchReceptors, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, OX-40, PAR2,PDGF-AA, PDGF-BB, PDGFRalpha, PDGFRbeta, PD-1, PD-L1, PD-L2,Phosphatidyl-serine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4,Sphingosine 1 Phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFbeta,TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFalpha,TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, Transferrin, Transferrin receptor,TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D,VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3.

In some embodiments, at least one antigen binding domain, orindependently each antigen binding domain, binds the tumor associatedantigen (TAA) folate receptor alpha (FRa). For example, the antigenbinding domain contains the binding domain as an sdAb that binds FRα.Exemplary FRα-binding sdAbs are set forth in SEQ ID NOS: 120, 121, and122.

In some embodiments, at least one antigen binding domain, orindependently each antigen binding domain, binds the tumor associatedantigen (TAA) cMET. For example, the antigen binding domain contains thebinding domain as a sdAb that binds cMET. An exemplary cMET-binding sdAbis set forth in SEQ ID NO: 123 (U.S. Pat. No. 9,346,884).

In some embodiments, at least one antigen binding domain, orindependently each antigen binding domain, binds the tumor associatedantigen (TAA) B7H3. For example, the antigen binding domain contains thebinding domain as an scFv that binds B7H3. An exemplary B7H3-bindingscFv is set forth in SEQ ID NO: 124. In some embodiments, the antigenbinding domain is a sdAb, such as a VHH. Exemplary B7H3-binding sdAbsare set forth in any of SEQ ID NOS: 214-218. In some embodiments, theantigen binding domain is or contains a Fab antibody fragment comprisinga VH-CH1 (Fd) and LC. An exemplary B7H3 Fd is set forth in SEQ ID NO:127 and an exemplary B7H3 LC is set forth in SEQ ID NO: 128 (PCTPublication No, WO2017/030926).

In some embodiments, at least one antigen binding domain, orindependently each antigen binding domain, binds the tumor associatedantigen (TAA) CD20. In some embodiments, such an antigen-binding domaincontains a VH set forth in SEQ ID NO: 189 and a VL set forth in SEQ IDNO: 190 or a sequence that exhibits at least at or about 85%, 90%, 95%,96%, 97%, 98%, 98%, or 99% sequence identity to SEQ ID NO: 189 or SEQ IDNO:190. For example, the antigen binding domain contains the bindingdomain as an scFv that binds CD20. Exemplary CD20-binding scFvs are setforth in SEQ ID NO: 125and 213 (U.S. Pub. No. US 2005/0123546).

In some embodiments, at least one antigen binding domain, orindependently each antigen binding domain, binds the tumor associatedantigen (TAA) DLL3. For example, the antigen binding domain contains thebinding domain as an scFv that binds DLL3. Exemplary DLL3-binding scFvis set forth in SEQ ID NO: 126 and 188 (U.S. Pub. No. US 2017/0037130).In some embodiments, the antigen binding domain is a sdAb, such as aVHH. Exemplary DLL3-binding sdAbs are set forth in any of SEQ ID NO: 219or SEQ ID NO:220. In some embodiments, the antigen binding domain is orcontains a Fab antibody fragment comprising a Fd and LC that binds DLL3.An exemplary DLL3 Fd is set forth in SEQ ID NO: 133 and an exemplaryDLL3 LC is set forth in SEQ ID NO: 134 (U.S. Pat. No. 8,044,178).

In some embodiments, at least one antigen binding domain, orindependently each antigen binding domain, binds the tumor associatedantigen (TAA) 5T4. An exemplary 5T4 Fd is set forth in SEQ ID NO: 129and an exemplary 5T4 LC is set forth in SEQ ID NO: 130. In someembodiments, the antibody binding domain comprises a VH-CH1 (Fd) orVL-CL as set forth in SEQ ID NOS: 167 and 168 (U.S. Pat. No. 8,044,178).

In some embodiments, at least one antigen binding domain, orindependently each antigen binding domain, binds the tumor associatedantigen (TAA) gpNMB. In some embodiments, the antigen binding domain isor contains a Fab fragment comprising a Fd and LC chain. An exemplarygpNMB Fd is set forth in SEQ ID NO: 131 and an exemplary gpNMB LC is setforth in SEQ ID NO: 132.

In some embodiments, the antigen binding domain is linked, directly orindirectly via a linker, to the Fc region and/or to the CD3 bindingregion. In some embodiments, linkage is via a linker. In someembodiments, the linker is a linking peptide (LP), which can include anyflexible or rigid linker as described in Section 11.3, althoughgenerally peptides linking the antigen binding domain or domains is nota cleavable linker.

In some embodiments, the multispecific polypeptide construct comprises afirst linking peptide (LP1) between the first antigen binding domain andthe Fc region. In some embodiments, the multispecific polypeptideconstruct comprises a second linking peptide (LP2) between the CD3binding region and the second antigen binding domain. In someembodiments, the multispecific polypeptide construct comprises a firstlinking peptide (LP1) between the first antigen binding domain and theFc region and a second linking peptide (LP2) between the CD3 bindingregion and the second antigen binding domain. In some aspects, themultispecific polypeptide construct has the structural arrangement fromN-terminus to C-terminus as follows: first antigen binding domain-LP1-Fcregion-linker-CD3 binding region-LP2-second antigen binding domain. Insome embodiments, the two linking peptides are not identical to eachother.

In some embodiments, the LP1 or LP2 is independently a peptide of about1 to 20 amino acids in length. In some embodiments, the LP1 or LP2 isindependently a peptide that is or comprises any Gly-Ser linker as setforth in SEQ ID NOs: 10-13, 119, 135, 147, 149 or GGS.

III. PHARMACEUTICAL COMPOSITION

Provided herein are compositions of any of the provided multispecificpolypeptide constructs. It will be appreciated that administration oftherapeutic entities in accordance with the disclosure will beadministered with suitable carriers, excipients, and other agents thatare incorporated into formulations to provide improved transfer,delivery, tolerance, and the like. A multitude of appropriateformulations can be found in the formulary known to all pharmaceuticalchemists: Remington's Pharmaceutical Sciences (15th ed., Mack PublishingCompany, Easton, Pa. (1975)), particularly Chapter 87 by Blaug, Seymour,therein. These formulations include, for example, powders, pastes,ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)containing vesicles (such as Lipofectin™), DNA conjugates, anhydrousabsorption pastes, oil-in-water and water-in-oil emulsions, emulsionscarbowax (polyethylene glycols of various molecular weights), semi-solidgels, and semi-solid mixtures containing carbowax. Any of the foregoingmixtures may be appropriate in treatments and therapies in accordancewith the present disclosure, provided that the active ingredient in theformulation is not inactivated by the formulation and the formulation isphysiologically compatible and tolerable with the route ofadministration. See also Baldrick P. “Pharmaceutical excipientdevelopment: the need for preclinical guidance.” Regul. ToxicolPharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and developmentof solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000),Charman WN “Lipids, lipophilic drugs, and oral drug delivery-someemerging concepts.” J Pharm Sci.89(8):967-78 (2000), Powell et al.“Compendium of excipients for parenteral formulations” PDA J Pharm SciTechnol. 52:238-311 (1998) and the citations therein for additionalinformation related to formulations, excipients and carriers well knownto pharmaceutical chemists.

In some embodiments, the multispecific polypeptide constructs,conjugated multispecific polypeptide constructs, and compositionsthereof—referred to collectively herein as the Therapeutic(s) andderivatives, fragments, analogs and homologs thereof, can beincorporated into pharmaceutical compositions suitable foradministration. Principles and considerations involved in preparing suchcompositions, as well as guidance in the choice of components areprovided, for example, in Remington's Pharmaceutical Sciences: TheScience And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al.,editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement:Concepts, Possibilities, Limitations, And Trends, Harwood AcademicPublishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery(Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

Such compositions typically comprise the multispecific polypeptideconstruct or a conjugated thereof and a pharmaceutically acceptablecarrier. Where a multispecific polypeptide construct includes a fragmentof an antibody, the smallest fragment of the antibody that specificallybinds to the target protein can be used. For example, based upon thevariable-region sequences of an antibody, peptide molecules can bedesigned that retain the ability of the antibody to bind the targetprotein sequence. Such peptides can be synthesized chemically and/orproduced by recombinant DNA technology. (See, e.g., Marasco et al.,Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)).

As used herein, the term “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Suitable carriers are described in the most recent edition ofRemington's Pharmaceutical Sciences, a standard reference text in thefield, which is incorporated herein by reference. Suitable examples ofsuch carriers or diluents include, but are not limited to, water,saline, ringer's solutions, dextrose solution, and 5% human serumalbumin. Liposomes and non-aqueous vehicles such as fixed oils may alsobe used. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe compositions is contemplated.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

A pharmaceutical composition of the disclosure is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfate; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be suitable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. Where the composition is lyophilized, sterilizationusing this method may be conducted either prior to or followinglyophilization and reconstitution. The composition for parenteraladministration may be stored in lyophilized form or in solution. Inaddition, parenteral compositions generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

In some embodiments, the pharmaceutical composition is administered to asubject through any route, including orally, transdermally, byinhalation, intravenously, intra-arterially, intramuscularly, directapplication to a wound site, application to a surgical site,intraperitoneally, by suppository, subcutaneously, intradermally,transcutaneously, by nebulization, intrapleurally, intraventricularly,intra-articularly, intraocularly, or intraspinally.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the multispecific polypeptideconstruct are delivered in the form of an aerosol spray from pressuredcontainer or dispenser that contains a suitable propellant, e.g., a gassuch as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the Therapeutics are prepared with carriers that willprotect the compound against rapid elimination from the body, such assustained/controlled release formulations, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art.

For example, the Therapeutics can be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions.

In some embodiments, the pharmaceutical composition comprises apharmaceutically-acceptable excipient, for example a filler, binder,coating, preservative, lubricant, flavoring agent, sweetening agent,coloring agent, a solvent, a buffering agent, a chelating agent, orstabilizer. Examples of pharmaceutically-acceptable fillers includecellulose, dibasic calcium phosphate, calcium carbonate,microcrystalline cellulose, sucrose, lactose, glucose, mannitol,sorbitol, maltol, pregelatinized starch, corn starch, or potato starch.Examples of pharmaceutically-acceptable binders includepolyvinylpyrrolidone, starch, lactose, xylitol, sorbitol, maltitol,gelatin, sucrose, polyethylene glycol, methyl cellulose, or cellulose.Examples of pharmaceutically-acceptable coatings include hydroxypropylmethylcellulose (HPMC), shellac, corn protein zein, or gelatin. Examplesof pharmaceutically-acceptable disintegrants includepolyvinylpyrrolidone, carboxymethyl cellulose, or sodium starchglycolate. Examples of pharmaceutically-acceptable lubricants includepolyethylene glycol, magnesium stearate, or stearic acid. Examples ofpharmaceutically-acceptable preservatives include methyl parabens, ethylparabens, propyl paraben, benzoic acid, or sorbic acid. Examples ofpharmaceutically-acceptable sweetening agents include sucrose,saccharine, aspartame, or sorbitol. Examples ofpharmaceutically-acceptable buffering agents include carbonates,citrates, gluconates, acetates, phosphates, or tartrates.

Sustained-release preparations can be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. In someembodiments, the pharmaceutical composition further comprises an agentfor the controlled or sustained release of the product, such asinjectable microspheres, bio-erodible particles, polymeric compounds(polylactic acid, polyglycolic acid), beads, or liposomes. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

The materials can also be obtained commercially from Alza Corporationand Nova Pharmaceuticals, Inc. Liposomal suspensions (includingliposomes targeted to infected cells with monoclonal antibodies to viralantigens) and can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the disclosure are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Further provided are kits comprising the pharmaceutical compositions (orarticles of manufacture) described herein. The pharmaceuticalcompositions can be included in a container, pack, or dispenser togetherwith instructions for administration. The kits described herein may alsoinclude other materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, syringes, andpackage inserts with instructions for performing any methods describedherein.

The formulation can also contain more than one multispecific polypeptideconstruct as necessary for the particular indication being treated, forexample, those with complementary activities that do not adverselyaffect each other. In some embodiments, or in addition, the compositioncan comprise an agent that enhances its function, such as, for example,a cytotoxic agent, cytokine, chemotherapeutic agent, orgrowth-inhibitory agent. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

In some embodiments, the dosage of the pharmaceutical composition is asingle dose or a repeated dose. In some embodiments, the doses are givento a subject once per day, twice per day, three times per day, or fouror more times per day. In some embodiments, about 1 or more (such asabout 2 or more, about 3 or more, about 4 or more, about 5 or more,about 6 or more, or about 7 or more) doses are given in a week. In someembodiments, multiple doses are given over the course of days, weeks,months, or years. In some embodiments, a course of treatment is about 1or more doses (such as about 2 or more does, about 3 or more doses,about 4 or more doses, about 5 or more doses, about 7 or more doses,about 10 or more doses, about 15 or more doses, about 25 or more doses,about 40 or more doses, about 50 or more doses, or about 100 or moredoses).

In some embodiments, the pharmaceutical composition is administered to asubject. Generally, dosages and routes of administration of thepharmaceutical composition are determined according to the size andcondition of the subject, according to standard pharmaceutical practice.For example, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in animal models such asmice, rats, rabbits, dogs, pigs, or monkeys. An animal model may also beused to determine the appropriate concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans. The exact dosage will bedetermined in light of factors related to the subject requiringtreatment. Dosage and administration are adjusted to provide sufficientlevels of the active compound or to maintain the desired effect. Factorsthat may be taken into account include the severity of the diseasestate, the general health of the subject, the age, weight, and gender ofthe subject, time and frequency of administration, drug combination(s),reaction sensitivities, and response to therapy. The optimal dosage andtreatment regime for a particular patient can readily be determined byone skilled in the art of medicine by monitoring the patient for signsof disease and adjusting the treatment accordingly.

IV. METHODS OF USE AND THERAPEUTIC ADMINISTRATION

Also provided are methods for using and uses of the multispecificpolypeptide constructs. Such methods and uses include therapeuticmethods and uses, for example, involving administration of the moleculesor compositions containing the same, to a subject having a disease,condition, or disorder, such as a tumor or cancer. In some embodiments,the molecule and/or composition is administered in an effective amountto effect treatment of the disease or disorder. Uses include uses of themultispecific polypeptide constructs in such methods and treatments, andin the preparation of a medicament in order to carry out suchtherapeutic methods. In some embodiments, the methods are carried out byadministering the multispecific polypeptide constructs, or compositionscomprising the same, to the subject having or suspected of having thedisease or condition. In some embodiments, the methods thereby treat thedisease or condition or disorder in the subject.

In one embodiment, a multispecific polypeptide construct of thedisclosure may be used as therapeutic agents. Such agents will generallybe employed to diagnose, prognose, monitor, treat, alleviate, and/orprevent a disease or pathology in a subject. A therapeutic regimen iscarried out by identifying a subject, e.g., a human patient or othermammal suffering from (or at risk of developing) a disorder usingstandard methods. A multispecific polypeptide construct is administeredto the subject. A multispecific polypeptide construct is administered tothe subject and will generally have an effect due to its binding withthe target(s).

In some embodiments, provided herein is a method of modulating an immuneresponse in a subject by administering a therapeutically effectiveamount of any of the provided multispecific conjugates or pharmaceuticalcompositions. In some embodiments, the method of modulating an immuneresponse increases or enhances an immune response in a subject. Forexample, the increase or enhanced response may be an increase incell-mediated immunity. In some examples, the method increases T-cellactivity, such as cytolytic T-cell (CTL) activity. In some embodiments,the modulated (e.g., increased) immune response is against a tumor orcancer.

Administration of the multispecific polypeptide construct may activateinnate immune cells via engagement of FcγRs through the Fc-region of themultispecific polypeptide construct. Administration of the multispecificpolypeptide construct may agonize, stimulate, activate, and/or augmentinnate immune cell effector functions, including ADCC, cytokine release,degranulation and/or ADCP. In examples where a multispecific polypeptideconstruct contains a cleavable linker, administration of themultispecific polypeptide construct may activate T-cell once thelinker(s) joining the first and second component is cleaved by aprotease thereby allowing the anti-CD3 binding portion to bind CD3ε onthe T cells. Administration of the multispecific polypeptide constructmay agonize, stimulate, activate, and/or augment CD3-mediated T cellactivation, cytotoxicity, cytokine release and/or proliferation.

In some embodiments, the provided methods are for treating a disease orcondition in a subject by administering a therapeutically effectiveamount of any of the provided multispecific conjugates or pharmaceuticalcompositions. In some embodiments, the disease or condition is a tumoror a cancer. Generally, alleviation or treatment of a disease ordisorder involves the lessening of one or more symptoms or medicalproblems associated with the disease or disorder. For example, in thecase of cancer, the therapeutically effective amount of the drug canaccomplish one or a combination of the following: reduce the number ofcancer cells; reduce the tumor size; inhibit (i.e., to decrease to someextent and/or stop) cancer cell infiltration into peripheral organs;inhibit tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. In some embodiments, a composition of this disclosure can beused to prevent the onset or reoccurrence of the disease or disorder ina subject, e.g., a human or other mammal, such as a non-human primate,companion animal (e.g., cat, dog, horse), farm animal, work animal, orzoo animal. The terms subject and patient are used interchangeablyherein.

In some embodiments, the pharmaceutical composition can be used toinhibit growth of mammalian cancer cells (such as human cancer cells). Amethod of treating cancer can include administering an effective amountof any of the pharmaceutical compositions described herein to a subjectwith cancer. The effective amount of the pharmaceutical composition canbe administered to inhibit, halt, or reverse progression of cancers.Human cancer cells can be treated in vivo, or ex vivo. In ex vivotreatment of a human patient, tissue or fluids containing cancer cellsare treated outside the body and then the tissue or fluids arereintroduced back into the patient. In some embodiments, the cancer istreated in a human patient in vivo by administration of the therapeuticcomposition into the patient.

Non-liming examples of disease include: all types of cancers (breast,lung, colorectal, prostate, melanomas, head and neck, pancreatic, etc.),rheumatoid arthritis, Crohn's disuse, SLE, cardiovascular damage,ischemia, etc. For example, indications would include leukemias,including T-cell acute lymphoblastic leukemia (T-ALL), lymphoblasticdiseases including multiple myeloma, and solid tumors, including lung,colorectal, prostate, pancreatic, and breast, including triple negativebreast cancer. For example, indications include bone disease ormetastasis in cancer, regardless of primary tumor origin; breast cancer,including by way of non-limiting example, ER/PR+ breast cancer, Her2+breast cancer, triple-negative breast cancer; colorectal cancer;endometrial cancer; gastric cancer; glioblastoma; head and neck cancer,such as esophageal cancer; lung cancer, such as by way of non-limitingexample, non-small cell lung cancer; multiple myeloma ovarian cancer;pancreatic cancer; prostate cancer; sarcoma, such as osteosarcoma; renalcancer, such as by way of nonlimiting example, renal cell carcinoma;and/or skin cancer, such as by way of nonlimiting example, squamous cellcancer, basal cell carcinoma, or melanoma. In some embodiments, thecancer is a squamous cell cancer. In some embodiments, the cancer is askin squamous cell carcinoma. In some embodiments, the cancer is anesophageal squamous cell carcinoma. In some embodiments, the cancer is ahead and neck squamous cell carcinoma. In some embodiments, the canceris a lung squamous cell carcinoma.

A therapeutically effective amount of a multispecific polypeptideconstruct of the disclosure relates generally to the amount needed toachieve a therapeutic objective. As noted above, this may be a bindinginteraction between the multispecific polypeptide construct and itstarget antigen(s) that, in certain cases, agonize, stimulate, activate,and/or augment FcγR-mediated innate immune cell activation orCD3-mediated T cell activation. The amount required to be administeredwill furthermore depend on the binding affinity of the multispecificpolypeptide construct for its specific antigen(s), and will also dependon the rate at which an administered multispecific polypeptide constructis depleted from the free volume other subject to which it isadministered. Common ranges for therapeutically effective dosing of amultispecific polypeptide construct may be, by way of nonlimitingexample, from about 0.01 μg/kg body weight to about 10 mg/kg bodyweight. In some embodiments, the therapeutically effective dosing of amultispecific polypeptide construct of the disclosure may be, by way ofnonlimiting example, from about 0.01 mg/kg body weight to about 5-10mg/kg body weight. Common dosing frequencies may range, for example,from twice daily to once a week.

Efficaciousness of treatment is determined in association with any knownmethod for diagnosing or treating the particular disorder. Methods forthe screening of multispecific polypeptide construct that possess thedesired specificity include, but are not limited to, enzyme linkedimmunosorbent assay (ELISA) and other immunologically mediatedtechniques known within the art. A variety of means are known fordetermining if administration of the provided multispecific polypeptideconstructs sufficiently modulates immunological activity by eliminating,sequestering, or inactivating immune cells mediating or capable ofmediating an undesired immune response; inducing, generating, or turningon immune cells that mediate or are capable of mediating a protectiveimmune response; changing the physical or functional properties ofimmune cells; or a combination of these effects. Examples ofmeasurements of the modulation of immunological activity include, butare not limited to, examination of the presence or absence of immunecell populations (using flow cytometry, immunohistochemistry, histology,electron microscopy, polymerase chain reaction (PCR)); measurement ofthe functional capacity of immune cells including ability or resistanceto proliferate or divide in response to a signal (such as using T-cellproliferation assays and pepscan analysis based on 3H-thymidineincorporation following stimulation with anti-CD3 antibody, anti-T-cellreceptor antibody, anti-CD28 antibody, calcium ionophores, PMA (phorbol12-myristate 13-acetate) antigen presenting cells loaded with a peptideor protein antigen; B cell proliferation assays); measurement of theability to kill or lyse other cells (such as cytotoxic T cell assays);measurements of the cytokines, chemokines, cell surface molecules,antibodies and other products of the cells (e.g., by flow cytometry,enzyme-linked immunosorbent assays, Western blot analysis, proteinmicroarray analysis, immunoprecipitation analysis); measurement ofbiochemical markers of activation of immune cells or signaling pathwayswithin immune cells (e.g., Western blot and immunoprecipitation analysisof tyrosine, serine or threonine phosphorylation, polypeptide cleavage,and formation or dissociation of protein complexes; protein arrayanalysis; DNA transcriptional, profiling using DNA arrays or subtractivehybridization); measurements of cell death by apoptosis, necrosis, orother mechanisms (e.g., annexin V staining, TUNEL assays, gelelectrophoresis to measure DNA laddering, histology; fluorogenic caspaseassays, Western blot analysis of caspase substrates); measurement of thegenes, proteins, and other molecules produced by immune cells (e.g.,Northern blot analysis, polymerase chain reaction, DNA microarrays,protein microarrays, 2-dimensional gel electrophoresis, Western blotanalysis, enzyme linked immunosorbent assays, flow cytometry); andmeasurement of clinical symptoms or outcomes such as improvement ofautoimmune, neurodegenerative, and other diseases involvingself-proteins or self-polypeptides (clinical scores, requirements foruse of additional therapies, functional status, imaging studies) forexample, by measuring relapse rate or disease severity.

The multispecific polypeptide construct are also useful in a variety ofdiagnostic and prophylactic formulations. In one embodiment, amultispecific polypeptide construct is administered to patients that areat risk of developing one or more of the aforementioned disorders. Apatient's or organ's predisposition to one or more of the disorders canbe determined using genotypic, serological or biochemical markers.

In another embodiment of the disclosure, a multispecific polypeptideconstruct is administered to human individuals diagnosed with a clinicalindication associated with one or more of the aforementioned disorders.Upon diagnosis, a multispecific polypeptide construct is administered tomitigate or reverse the effects of the clinical indication.

Combination Therapies

In some embodiments, the multispecific polypeptide constructs,conjugated multispecific polypeptide constructs, and compositionsthereof—referred to collectively herein as the Therapeutic(s)—areadministered in conjunction with one or more additional agents, or acombination of additional agents. Suitable additional agents includecurrent pharmaceutical and/or surgical therapies for an intendedapplication. For example, the Therapeutic(s) can be used in conjunctionwith an additional chemotherapeutic or anti-neoplastic agent. Forexample, the Therapeutic(s) and additional agent are formulated into asingle therapeutic composition, and the Therapeutic(s) and additionalagent are administered simultaneously. In some embodiments, theTherapeutic(s) and additional agent are separate from each other, e.g.,each is formulated into a separate therapeutic composition, and theTherapeutic(s) and the additional agent are administered simultaneously,or the Therapeutic(s) and the additional agent are administered atdifferent times during a treatment regimen. For example, theTherapeutic(s) is administered prior to the administration of theadditional agent, the Therapeutic(s) is administered subsequent to theadministration of the additional agent, or the Therapeutic(s) and theadditional agent are administered in an alternating fashion. Asdescribed herein, the Therapeutic(s) and additional agent areadministered in single doses or in multiple doses. In some embodiments,the additional agent is coupled or otherwise attached to theTherapeutic(s). Suitable additional agents are selected according to thepurpose of the intended application (i.e., killing, prevention of cellproliferation, hormone therapy or gene therapy). Such agents may includebut is not limited to, for example, pharmaceutical agents, toxins,fragments of toxins, alkylating agents, enzymes, antibiotics,antimetabolites, antiproliferative agents, hormones, neurotransmitters,DNA, RNA, siRNA, oligonucleotides, antisense RNA, aptamers, diagnostics,radiopaque dyes, radioactive isotopes, fluorogenic compounds, magneticlabels, nanoparticles, marker compounds, lectins, compounds that altercell membrane permeability, photochemical compounds, small molecules,liposomes, micelles, gene therapy vectors, viral vectors, and the like.Finally, combinations of agents or combinations of different classes ofagents may be used.

In one embodiment, the multispecific polypeptide constructs areadministered in combination therapy, i.e., combined with other agents,e.g., therapeutic agents, that are useful for treating pathologicalconditions or disorders, such as autoimmune disorders and inflammatorydiseases. The term “in combination” in this context means that theagents are given substantially contemporaneously, either simultaneouslyor sequentially. If given sequentially, at the onset of administrationof the second compound, the first of the two compounds is stilldetectable at effective concentrations at the site of treatment.

For example, the combination therapy can include one or moremultispecific polypeptide constructs of the disclosure co-formulatedwith, and/or co-administered with, one or more additional therapeuticagents, e.g., one or more cytokine and growth factor inhibitors,immunosuppressants, anti-inflammatory agents, metabolic inhibitors,enzyme inhibitors, and/or cytotoxic or cytostatic agents, as describedin more detail below. Furthermore, one or more multispecific polypeptideconstructs described herein may be used in combination with two or moreof the therapeutic agents described herein. Such combination therapiesmay advantageously utilize lower dosages of the administered therapeuticagents, thus avoiding possible toxicities or complications associatedwith the various monotherapies.

In other embodiments, one or more multispecific polypeptide constructsof the disclosure can be co-formulated with, and/or co-administeredwith, one or more anti-inflammatory drugs, immunosuppressants, ormetabolic or enzymatic inhibitors. Nonlimiting examples of the drugs orinhibitors that can be used in combination with the antibodies describedherein, include, but are not limited to, one or more of: nonsteroidalanti-inflammatory drug(s) (NSAIDs), e.g., ibuprofen, tenidap, naproxen,meloxicam, piroxicam, diclofenac, and indomethacin; sulfasalazine;corticosteroids such as prednisolone; cytokine suppressiveanti-inflammatory drug(s) (CSAIDs); inhibitors of nucleotidebiosynthesis, e.g., inhibitors of purine biosynthesis, folateantagonists (e.g., methotrexate(N-[4-[[(2,4-diamino-6-pteridinyl)methyl] methylamino]benzoyl]-L-glutamic acid); and inhibitors of pyrimidine biosynthesis,e.g., dihydroorotate dehydrogenase (DHODH) inhibitors. Suitabletherapeutic agents for use in combination with the antibodies of thedisclosure include NSAIDs, CSAIDs, (DHODH) inhibitors (e.g.,leflunomide), and folate antagonists (e.g., methotrexate).

Examples of additional inhibitors include one or more of:corticosteroids (oral, inhaled and local injection); immunosuppressants,e.g., cyclosporin, tacrolimus (FK-506); and mTOR inhibitors, e.g.,sirolimus (rapamycin—RAPAMUNE™ or rapamycin derivatives, e.g., solublerapamycin derivatives (e.g., ester rapamycin derivatives, e.g.,CCI-779); agents that interfere with signaling by proinflammatorycytokines such as TNFα or IL-1 (e.g. IRAK, NIK, IKK, p38 or MAP kinaseinhibitors); COX2 inhibitors, e.g., celecoxib, rofecoxib, and variantsthereof; phosphodiesterase inhibitors, e.g., R973401 (phosphodiesteraseType IV inhibitor); phospholipase inhibitors, e.g., inhibitors ofcytosolic phospholipase 2 (cPLA2) (e.g., trifluoromethyl ketoneanalogs); inhibitors of vascular endothelial cell growth factor orgrowth factor receptor, e.g., VEGF inhibitor and/or VEGF-R inhibitor;and inhibitors of angiogenesis. Suitable therapeutic agents for use incombination with the antibodies of the disclosure areimmunosuppressants, e.g., cyclosporin, tacrolimus (FK-506); mTORinhibitors, e.g., sirolimus (rapamycin) or rapamycin derivatives, e.g.,soluble rapamycin derivatives (e.g., ester rapamycin derivatives, e.g.,CCI-779); COX2 inhibitors, e.g., celecoxib and variants thereof; andphospholipase inhibitors, e.g., inhibitors of cytosolic phospholipase 2(cPLA2), e.g., trifluoromethyl ketone analogs. Additional examples oftherapeutic agents that can be combined with a multispecific polypeptideconstruct include one or more of: 6-mercaptopurines (6-MP); azathioprinesulphasalazine; mesalazine; olsalazine; chloroquine/hydroxychloroquine(PLAQUENIL®); pencillamine; aurothiornalate (intramuscular and oral);azathioprine; coichicine; beta-2 adrenoreceptor agonists (salbutamol,terbutaline, salmeteral); xanthines (theophylline, aminophylline);cromoglycate; nedocromil; ketotifen; ipratropium and oxitropium;mycophenolate mofetil; adenosine agonists; antithrombotic agents;complement inhibitors; and adrenergic agents.

V. EXEMPLARY EMBODIMENTS

Among the provided embodiments are:

1. A multispecific polypeptide construct, the multispecific polypeptideconstruct comprising a first component comprising an immunoglobulin Fcregion and a second component comprising a CD3-binding region, wherein:

the first and second components are coupled by a non-cleavable linker,wherein the Fc region is positioned N-terminal to the CD3-bindingregion; and

one or both of the first and second components comprises an antigenbinding domain that binds a tumor associated antigen (TAA).

2. The multispecific polypeptide construct of embodiment 1, wherein theCD3-binding region binds CD3 (CD3ε).

3. The multispecific construct of embodiment 1 or embodiment 2, whereinthe antigen binding domain is positioned amino-terminally relative tothe Fc region and/or carboxy-terminally relative to the CD3 bindingregion of the multispecific polypeptide construct.

4. The multispecific polypeptide construct of any of embodiments 1-3,wherein the first component comprises a first antigen binding domain andthe second component comprises a second antigen binding domain, whereineach of the antigen binding domains bind a tumor associated antigen(TAA).

5. The multispecific polypeptide construct of embodiment 4, wherein thefirst antigen binding domain is positioned amino-terminally relative tothe Fc region of the multispecific construct and the second antigenbinding domain is positioned carboxy-terminally relative to the CD3binding region of the multispecific construct.

6. A multispecific polypeptide construct, wherein the multispecificconstruct comprises in order, from N-terminus to C-terminus:

a first antigen binding domain that binds to a tumor-associated antigen(TAA);

an immunoglobulin Fc region;

a non-cleavable linker;

a CD3 binding region that binds CD3 (CD3ε); and

a second antigen binding domain that binds a tumor-associated antigen(TAA).

7. A multispecific polypeptide construct, wherein the multispecificconstruct comprises in order, from N-terminus to C-terminus:

an immunoglobulin Fc region;

a non-cleavable linker;

a CD3 binding region that binds CD3 (CD3ε); and

an antigen binding domain that binds a tumor-associated antigen (TAA).

8. A multispecific polypeptide construct, wherein the multispecificconstruct comprises in order, from N-terminus to C-terminus:

an antigen binding domain that binds to a tumor-associated antigen(TAA);

an immunoglobulin Fc region;

a non-cleavable linker; and

a CD3 binding region that binds CD3 (CD3ε).

9. The multispecific polypeptide construct of any of embodiments 1-8,wherein the Fc region is a homodimeric Fc region.

10. The multispecific polypeptide construct of any of embodiments 1-9,wherein the Fc region is an Fc region of a human IgG1, a human IgG2, ahuman IgG3, or a human IgG4, or is an immunologically active fragmentthereof.

11. The multispecific polypeptide construct of any of embodiments 1-10,wherein the Fc region comprises a polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 1 or a sequence of amino acids that hasat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequenceidentity to SEQ ID NO:1.

12. The multispecific polypeptide construct of any of embodiments 1-10,wherein the Fc region comprises a polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 2 or a sequence of amino acids that hasat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequenceidentity to SEQ ID NO:2;

the Fc region comprises a polypeptide comprises the amino acid sequenceset forth in SEQ ID NO: 4 or a sequence of amino acids that has at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity toSEQ ID NO:4; or

the Fc region comprises a polypeptide comprises the amino acid sequenceset forth in SEQ ID NO: 5 or a sequence of amino acids that has at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity toSEQ ID NO:5.

13. The multispecific polypeptide construct of any of embodiments 1-6, 9and 12, wherein the Fc region is a heterodimeric Fc region.

14. The multispecific polypeptide construct of embodiment 13, whereinone or both Fc polypeptides of the heterodimeric Fc region comprises atleast one modification to induce heterodimerization compared to apolypeptide of a homodimeric Fc region, optionally compared to the Fcpolypeptide set forth in SEQ ID NO:1 or an immunologically activefragment thereof.

15. The multispecific polypeptide construct of embodiment 14, whereineach of the Fc polypeptides of the heterodimeric Fc independentlycomprise at least one amino acid modification.

16. The multispecific polypeptide construct of embodiment 15, whereineach of the Fc polypeptides of the heterodimeric Fc comprise aknob-into-hole modification or comprise a charge mutation to increaseelectrostatic complementarity of the polypeptides.

17. The multispecific polypeptide construct of embodiment 16, whereinthe amino acid modification is a knob-into-hole modification.

18. The multispecific polypeptide construct of any of embodiments 13-17,wherein the first Fc polypeptide of the heterodimeric Fc comprises themodification selected from among Thr366Ser, Leu368Ala, Tyr407Val, andcombinations thereof and the second Fc polypeptide of the heterodimericFc comprises the modification T366W.

19. The multispecific polypeptide construct of embodiment 18, whereinthe first and second Fc polypeptides further comprises a modification ofa non-cysteine residue to a cysteine residue, wherein the modificationof the first polypeptide is at one of the position Ser354 and Y349 andthe modification of the second Fc polypeptide is at the other of theposition Ser354 and Y349.

20. The multispecific polypeptide construct of embodiment 16, whereinthe amino acid modification is a charge mutation to increaseelectrostatic complementarity of the polypeptides.

21. The multispecific polypeptide construct of any of embodiments 13-16and 20, wherein the first and/or second Fc polypeptides or each of thefirst and second Fc polypeptide comprise a modification in complementarypositions, wherein the modification is replacement with an amino acidhaving an opposite charge to the complementary amino acid of the otherpolypeptide.

22. The multispecific polypeptide construct of any of embodiments 14-21,wherein one of the first or second Fc polypeptide of the heterodimericFc further comprises a modification at residue Ile253.

23. The multispecific polypeptide construct of embodiment 22, whereinthe modification is Ile253Arg.

24. The multispecific polypeptide construct of any of embodiments 14-23,wherein one of the first or second Fc polypeptide of the heterodimericFc further comprises a modification at residue His435.

25. The multispecific polypeptide construct of embodiment 24, whereinthe modification is His435Arg.

26. The multispecific polypeptide construct of any of embodiments 1-25,wherein the Fc region comprises a polypeptide that lacks Lys447.

27. The multispecific polypeptide construct of any of embodiments 1-26,wherein the Fc region comprises a polypeptide comprising at least onemodification to enhance FcRn binding.

28. The multispecific polypeptide construct of embodiment 27, whereinthe modification is at a position selected from the group consisting ofMet252, Ser254, Thr256, Met428, Asn434, and combinations thereof.

29. The multispecific polypeptide construct of embodiment 28, whereinthe modification is at a position selected from the group consisting ofMet252Y, Ser254T, Thr256E, Met428L, Met428V, Asn434S, and combinationsthereof.

30. The multispecific polypeptide construct of embodiment 28, whereinthe modification is at position Met252 and at position Met428.

31. The multispecific polypeptide construct of embodiment 30, whereinthe modification is Met252Y and Met428L.

32. The multispecific polypeptide construct of embodiment 30, whereinthe modification is Met252Y and Met428V.

33. The multispecific polypeptide construct of any of embodiments 13-32,wherein the first polypeptide of the heterodimeric Fc comprises thesequence of amino acids set forth in any of SEQ ID NOS:82, 86, 94 or 96,and the second polypeptide of the heterodimeric Fc comprises thesequence of amino acids set forth in any of SEQ ID NOS:83, 87, 90, 92,98 or 100.

34. The multispecific polypeptide construct of any of embodiments 1-33,wherein the Fc region comprises a polypeptide comprising at least oneamino acid modification that reduces effector function and/or reducesbinding to an effector molecule selected from an Fc gamma receptor orC1q.

35. The multispecific polypeptide construct of embodiment 34, whereinthe one or more amino acid modification is deletion of one or more ofGlu233, Leu234 or Leu235.

36. The multispecific polypeptide construct of any of embodiments 13-32,34 and 35, wherein the first polypeptide of the heterodimeric Fccomprises the sequence of amino acids set forth in any of SEQ ID NOS:84, 88, 95 or 97 and the second polypeptide of the heterodimeric Fccomprises the sequence of amino acids set forth in any of SEQ ID NOS:85, 89, 91, 93, 99 or 101.

37. The multispecific polypeptide construct of any of embodiments 1-32,wherein the Fc region comprises a polypeptide comprising at least onemodification to enhance FcγR binding.

38. The multispecific polypeptide construct of embodiment 37, whereinthe modification is modification at Ser239 or Ile332.

39. The multispecific polypeptide construct of any of embodiments 1-32and 37, wherein the glycosylation of the Fc region is modified toenhance Fc□R binding as compared to an unmodified Fc region.

40. The multispecific polypeptide construct of embodiment 39, whereinthe Fc region lacks or has reduced fucose content.

41. The multispecific polypeptide construct of any of embodiments 1-40,wherein the CD3 binding region is an anti-CD3 antibody orantigen-binding fragment.

42. The multispecific polypeptide construct of embodiment 41, whereinthe anti-CD3 antibody or antigen binding fragment comprises a variableheavy chain region (VH) and a variable light chain region (VL).

43. The multispecific polypeptide construct of any of embodiments 1-42,wherein the CD3 binding region is monovalent.

44. The multispecific polypeptide construct of any of embodiments 41-43,wherein the anti-CD3 antibody or antigen binding fragment is not asingle chain antibody, optionally is not a single chain variablefragment (scFv).

45. The multispecific polypeptide construct of embodiment 42 orembodiment 44, wherein the Fc is a heterodimeric Fc and the VH and VLthat comprise the anti-CD3 antibody or antigen binding fragment arelinked to opposite polypeptides of the heterodimeric Fc.

46. The multispecific polypeptide construct of any of embodiments 1-45,wherein the CD3 binding region is not able to, or is not substantiallyable to, bind or engage CD3 unless at least one of the antigen bindingdomain is bound to its TAA.

47. The multispecific polypeptide construct of any of embodiments 1-46,wherein the CD3 binding region is not able to, or is not substantiallyable, to bind or engage CD3 unless at least two of the antigen bindingdomain is bound to its TAA.

48. The multispecific polypeptide construct of any of embodiments 1-47,wherein the linker is a polypeptide linker.

49. The multispecific polypeptide construct of embodiment 48, whereinthe linker is a polypeptide of up to 25 amino acids in length.

50. The multispecific polypeptide construct of embodiment 48 orembodiment 49, wherein the linker is a polypeptide of from or from about2 to 24 amino acids, 2 to 20 amino acids, 2 to 18 amino acids, 2 to 14amino acids, 2 to 12 amino acids, 2 to 10 amino acids, 2 to 8 aminoacids, 2 to 6 amino acids, 6 to 24 amino acids, 6 to 20 amino acids, 6to 18 amino acids, 6 to 14 amino acids, 6 to 12 amino acids, 6 to 10amino acids, 6 to 8 amino acids, 8 to 24 amino acids, 8 to 20 aminoacids, 8 to 18 amino acids, 8 to 14 amino acids, 8 to 12 amino acids, 8to 10 amino acids, 10 to 24 amino acids, 10 to 20 amino acids, 10 to 18amino acids, 10 to 14 amino acids, 10 to 12 amino acids, 12 to 24 aminoacids, 12 to 20 amino acids, 12 to 18 amino acids, 12 to 14 amino acids,14 to 24 amino acids, 14 to 20 amino acids, 14 to 18 amino acids, 18 to24 amino acids, 18 to 20 amino acids or 20 to 24 amino acids.

51. The multispecific polypeptide construct of any of embodiments 48-50,wherein the linker is a polypeptide that is 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length.

52. The multispecific polypeptide construct of any of embodiments 48-51,wherein the linker is a polypeptide that is 3 to 18 amino acids inlength.

53. The multispecific polypeptide construct of any of embodiments 48-51,wherein the linker is a polypeptide that is 12 to 18 amino acids inlength.

54. The multispecific polypeptide construction of any of embodiments48-51, wherein the linker is a polypeptide that is 15 to 18 amino acidsin length.

55. A multispecific polypeptide construct, the multispecific polypeptideconstruct comprising a first component comprising a heterodimeric Fcregion and a second component comprising an anti-CD3 antibody orantigen-binding fragment comprising a variable heavy chain region (VH)and a variable light chain region (VL), wherein:

the VH and VL that comprise the anti-CD3 antibody or antigen bindingfragment are linked to opposite polypeptides of the heterodimeric Fc;

the first and second components are coupled by a non-cleavable linker,wherein the heterodimeric Fc region is positioned N-terminal to theanti-CD3 antibody; and

one or both of the first and second components comprises an antigenbinding domain that binds a tumor associated antigen (TAA).

56. The multispecific polypeptide construct of any of embodiments 52-55,wherein the non-cleavable linker does not contain a substraterecognition site that is specifically recognized for cleavage by aprotease.

57. The multispecific polypeptide construct of embodiment 56, whereinthe protease is produced by an immune effector cell, by a tumor, or bycells present in the tumor microenvironment.

58. The multispecific polypeptide construct of embodiment 57, whereinthe protease is produced by an immune effector cell and the immuneeffector cell is an activated T cell, a natural killer (NK) cell, or anNK T cell.

59. The multispecific polypeptide construct of any of embodiments 56-58,wherein the protease is selected from among matriptase, a matrixmetalloprotease (MMP), Granzyme B, and combinations thereof.

60. The multispecific polypeptide construct of embodiment 59, whereinthe protease is granzyme B.

61. The multispecific polypeptide construct of any of embodiments 1-60,wherein the non-cleavable linker comprises GS, GGS, GGGGS (SEQ IDNO:149), GGGGGS (SEQ ID NO:135) and combinations thereof.

62. The multispecific polypeptide construct of any of embodiments 1-61,wherein the non-cleavable linker comprises (GGS)n, wherein n is 1 to 10.

63. The multispecific polypeptide construct of any of embodiments 1-62,wherein the non-cleavable linker comprises (GGGGS)n (SEQ ID NO: 173),wherein n is 1 to 10.

64. The multispecific polypeptide construct of any of embodiments 1-62,wherein the non-cleavable linker comprises (GGGGGS)n (SEQ ID NO:172),wherein n is 1 to 4.

65. The multispecific polypeptide construct of any of embodiments 1-64,wherein the non-cleavable linker comprises GGS.

66. The multispecific polypeptide construct of any of embodiments 1-64,wherein the non-cleavable linker comprises GGGGS (SEQ ID NO: 149).

67. The multispecific polypeptide construct of any of embodiments 1-64,wherein the non-cleavable linker comprises GGGGGS (SEQ ID NO: 135).

68. The multispecific polypeptide construct of any of embodiments 1-64,wherein the non-cleavable linker comprises (GGS)2 (SEQ ID NO: 10).

69. The multispecific polypeptide construct of any of embodiments 1-64,wherein the non-cleavable linker comprises GGSGGSGGS (SEQ ID NO: 11).

70. The multispecific polypeptide construct of any of embodiments 1-64,wherein the non-cleavable linker comprises GGSGGSGGSGGS (SEQ ID NO: 12).

71. The multispecific polypeptide construct of any of embodiments 1-64,wherein the non-cleavable linker comprises GGSGGSGGSGGSGGS (SEQ ID NO:13).

72. The multispecific polypeptide construct of any of embodiments 1-64,wherein the non-cleavable linker comprises GGGGGSGGGGGSGGGGGS (SEQ IDNO: 119).

73. The multispecific polypeptide construct of any of embodiments 1-64,wherein the non-cleavable linker comprises GGSGGGGSGGGGSGGGGS (SEQ IDNO: 147)

74. The multispecific polypeptide construct of any of embodiments 1-64,wherein the non-cleavable linker comprises and GGGGSGGGGSGGGGS (SEQ IDNO:170).

75. The multispecific polypeptide construct of any of embodiments 45-74,wherein the multispecific polypeptide construct comprises at least (i) afirst polypeptide comprising the first Fc polypeptide of theheterodimeric Fc region, the linker and the VH domain of the anti-CD3antibody or antigen binding fragment; and (ii) a second polypeptidecomprising the second Fc polypeptide of the heterodimeric Fc region, thelinker and the VL domain of the anti-CD3 antibody or antigen bindingfragment, wherein one or both of the first and second polypeptidecomprise at least one antigen-binding domain that binds to a tumorassociated antigen (TAA).

76. The multispecific polypeptide construct of any of embodiments 1-75,wherein one or more antigen binding domain that binds TAA results inmonovalent, bivalent, trivalent, or tetravalent binding to the TAA.

77. The multispecific polypeptide construct of embodiment 75, whereinonly one of the first or second polypeptide comprises the at least oneantigen-binding domain that binds a TAA.

78. The multispecific polypeptide construct of embodiment 75 orembodiment 77, wherein the at least one antigen binding domain ispositioned amino-terminally relative to the Fc region and/or ispositioned carboxy-terminally relative to the CD3 binding region of oneof the first or second polypeptide of the multispecific polypeptideconstruct.

79. The multispecific polypeptide construct of embodiment 75 orembodiment 77, wherein the at least one antigen binding domain ispositioned amino-terminally relative to the Fc region of themultispecific construct and the second antigen binding domain ispositioned carboxy-terminally relative to the CD3 binding region of themultispecific construct.

80. The multispecific polypeptide construct of any of embodiments 1-79,wherein the antigen binding domain, or independently each of the antigenbinding domains, comprises an extracellular domain or binding fragmentthereof of the native cognate binding partner of the TAA, or a variantthereof that exhibits binding activity to the TAA.

81. The multispecific polypeptide construct of any of embodiments 1-79,wherein the antigen binding domain, or independently each of the antigenbinding domains, is an antibody or antigen-binding fragment thereofselected from the group consisting of a Fab fragment, a F(ab′)2fragment, an Fv fragment, a scFv, a scAb, a dAb, a single domain heavychain antibody, and a single domain light chain antibody.

82. The multispecific polypeptide construct of embodiment 81, whereinthe antibody or antigen-binding fragment thereof is a Fv, a scFv, a Fab,a single domain antibody (sdAb), a VNAR, or a VHH.

83. The multispecific polypeptide construct of embodiment 81 orembodiment 82, wherein the antibody or antigen-binding fragment is ansdAb.

84. The multispecific polypeptide construct of embodiment 83, whereinthe sdAb is a human or humanized sdAb.

85. The multispecific polypeptide construct of embodiment 83 orembodiment 84, wherein the sdAb is VHH, VNAR, an engineered VH domain oran engineered VK domain.

86. The multispecific polypeptide construct of embodiment 81 orembodiment 82, wherein the antibody or antigen-binding fragment thereofis an scFv.

87. The multispecific polypeptide construct of embodiment 81 orembodiment 82, wherein the antibody or antigen-binding fragment thereofis a Fab.

88. The multispecific polypeptide construct of embodiment 87, whereinthe multispecific polypeptide construct comprises:

(i) a first polypeptide comprising the first Fc polypeptide of theheterodimeric Fc region, the linker and the VH domain of the anti-CD3antibody or antigen binding fragment;

(ii) a second polypeptide comprising the second Fc polypeptide of theheterodimeric Fc region, the linker and the VL domain of the anti-CD3antibody or antigen binding fragment, and

(iii) a third polypeptide comprising a VH-CH1 (Fd) or VL-CL of a Fabantibody fragment that binds to a tumor-associated antigen, wherein thefirst and/or second polypeptide further comprises the other of theVH-CH1 (Fd) or VL-CL of the Fab antibody fragment.

89. The multispecific polypeptide construct of embodiment 88, whereinonly one of the first or second polypeptide comprises the other of theVH-CH1 (Fd) or VL-CL of the Fab antibody fragment.

90. The multispecific polypeptide construct of embodiment 89, whereinboth the first or second polypeptide comprises the other of the VH-CH1(Fd) or VL-CL of the Fab antibody fragment.

91. The multispecific polypeptide construct of embodiment 89 orembodiment 90, wherein the other of the VH-CH1 (Fd) or VL-CL of the Fabantibody fragment is positioned amino-terminally relative to the Fcregion and/or at the carboxy-terminally relative to the CD3 bindingregion of one of the first or second polypeptide of the multispecificpolypeptide construct.

92. The multispecific polypeptide construct of any of embodiments 89-91,wherein the other of the VH-CH1 (Fd) or VL-CL of the Fab antibodyfragment is positioned amino-terminally relative to the Fc region of thefirst polypeptide or second polypeptide and at the carboxy-terminallyrelative to the CD3 binding region of the other of the first or secondpolypeptide.

93. The multispecific polypeptide construct of any of embodiments 1-92,wherein the antigen binding domain, or independently each of the antigenbinding domains, binds to a tumor antigen selected from among1-92-LFA-3, 5T4, Alpha-4 integrin, Alpha-V integrin, alpha4beta1integrin, alpha4beta7 integrin, AGR2, Anti-Lewis-Y, Apelin J receptor,APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9,(Lewis a), Carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20,CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44,CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81,CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138,CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), CLAUDIN-3,CLAUDIN-4, cMet, Collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10,CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1,EDA, EDB, EGFR, EGFRviii, Endothelin B receptor (ETBR), ENPP3, EpCAM,EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2,FGFR3, FGFR4, FLT-3, Folate receptor alpha (FRα), GAL3ST1, G-CSF,G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GP IIb/IIIa receptors,Gp130, GPIIB/IIIA, GPNMB, GRP78, HER2/neu, HER3, HER4, HGF, hGH, HVEM,Hyaluronidase, ICOS, IFNalpha, IFNbeta, IFNgamma, IgE, IgE Receptor(FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R,IL-12Rbeta1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R,IL27/IL27R (wsx1), IL29, IL-31R, IL31/IL31R, IL2R, IL4, IL4R, IL6, IL6R,Insulin Receptor, Jagged Ligands, Jagged 1, Jagged 2, KISS1-R, LAG-3,LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, Mesothelin,MRP4, MUC1, Mucin-16 (MUC16, CA-125), Na/K ATPase, NGF, Nicastrin, NotchReceptors, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, OX-40, PAR2,PDGF-AA, PDGF-BB, PDGFRalpha, PDGFRbeta, PD-1, PD-L1, PD-L2,Phosphatidyl-serine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4,Sphingosine 1 Phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFbeta,TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFalpha,TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, Transferrin, Transferrin receptor,TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D,VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3.

94. The multispecific polypeptide construct of any of embodiments 1-93,wherein multispecific antigen binding domain comprises at least a firstantigen binding domain and a second antigen binding domain, wherein thefirst antigen binding domain and second antigen binding domain bind tothe same TAA.

95. The multispecific polypeptide construct of embodiment 94, whereinthe first antigen binding domain and the second antigen binding domainbinds a different epitope of the same TAA.

96. The multispecific polypeptide construct of embodiment 94, whereinthe first antigen binding domain and the second antigen binding domainbinds the same epitope of the same TAA.

97. The multispecific polypeptide construct of any of embodiments 1-96,wherein multispecific antigen binding domain comprises at least a firstantigen binding domain and a second antigen binding domain wherein thefirst antigen binding domain and the second antigen binding domain binda different TAA.

98. The multispecific polypeptide construct of any of embodiments 5-97,wherein the multispecific polypeptide construct comprises a firstlinking peptide (LP1) between the first antigen binding domain and theFc region.

99. The multispecific polypeptide construct of any of embodiments 5-98,wherein the multispecific polypeptide construct comprises a secondlinking peptide (LP2) between the CD3 binding region and the secondantigen binding domain.

100. The multispecific polypeptide construct of any of embodiments 5-99,wherein the multispecific polypeptide construct comprises a firstlinking peptide (LP1) between the first antigen binding domain and theFc region and a second linking peptide (LP2) between the CD3 bindingregion and the second antigen binding domain, and wherein themultispecific polypeptide construct has the structural arrangement fromN-terminus to C-terminus as follows: first antigen binding domain-LP1-Fcregion-non-cleavable linker-CD3 binding region-LP2-second antigenbinding domain.

101. The multispecific polypeptide construct of embodiment 100, whereinthe two linking peptides are identical to each other.

102. The multispecific polypeptide construct of embodiment 100, whereinthe two linking peptides are not identical to each other.

103. The multispecific polypeptide construct of any of embodiments98-102, wherein LP1 or LP2 is independently a peptide of about 1 to 20amino acids in length.

104. The multispecific polypeptide of embodiment 103, wherein LP1 or LP2independently comprise a peptide that is or comprises any Gly-Ser linkeras set forth in SEQ ID NOs: 10-13, 119, 135, 147, 149 or GGS.

105. The multispecific polypeptide construct of any of embodiments41-104, wherein the anti-CD3 antibody or antigen binding fragment is anFv antibody fragment.

106. The multispecific polypeptide construct of embodiment 105, whereinthe Fv antibody fragment comprises a disulfide stabilized anti-CD3binding Fv fragment (dsFv).

107. The multispecific polypeptide construct of any of embodiments41-106, wherein:

the anti-CD3 antibody or antigen-binding fragment comprises a VH CDR1comprising the amino acid sequence TYAMN (SEQ ID NO: 16) or SEQ IDNO:211; a VH CD2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD(SEQ ID NO: 17) or SEQ ID NO:212; a VH CDR3 comprising the amino acidsequence HGNFGNSYVSWFAY (SEQ ID NO: 18), a VL CDR1 comprising the aminoacid sequence RSSTGAVTTSNYAN (SEQ ID NO: 19); a VL CDR2 comprising theamino acid sequence GTNKRAP (SEQ ID NO: 20); and a VL CDR3 comprisingthe amino acid sequence ALWYSNLWV (SEQ ID NO: 21): or the anti-CD3antibody or antigen-binding fragment comprises a VH CDR1 sequence thatincludes at least the amino acid sequence GFTFNTYAMN (SEQ ID NO: 211); aVH CDR2 sequence that includes at least the amino acid sequenceRIRSKYNNYATY (SEQ ID NO: 212); a VH CDR3 sequence that includes at leastthe amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 18), a VL CDR1sequence that includes at least the amino acid sequence GSSTGAVTTSNYAN(SEQ ID NO: 229); a VL CDR2 sequence that includes at least the aminoacid sequence GTNKRAP (SEQ ID NO: 230); and a VL CDR3 sequence thatincludes at least the amino acid sequence ALWYSNHWV (SEQ ID NO: 225).

108. The multispecific polypeptide construct of embodiment 106 orembodiment 107, wherein the anti-CD3 dsFv comprises:

a VH having the amino acid sequence of any of SEQ ID NOS: 14 and 32-62or a sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% sequence identity to any of SEQ ID NOS: 14 and 32-62;and

a VL having the amino acid sequence of any of SEQ ID NOS: 15 and 63-81or a sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% sequence identity to any of SEQ ID NOS: 14 and 32-62.

109. The multispecific polypeptide construct of any of embodiments106-108, wherein the anti-CD3 dsFv comprises the amino acid sequence ofSEQ ID NO: 14 and the amino acid sequence of SEQ ID NO: 15.

110. The multispecific polypeptide construct of any of embodiments102-104, wherein the anti-CD3 dsFv comprises the amino acid sequence ofSEQ ID NO: 44 and the amino acid sequence of SEQ ID NO: 72.

111. The multispecific polypeptide construct of any of embodiments1-109, wherein the multispecific polypeptide construct is conjugated toan agent.

112. The multispecific polypeptide construct of embodiment 111, whereinthe agent is a therapeutic agent, an antineoplastic agent, a toxin orfragment thereof, a detectable moiety or a diagnostic agent.

113. The multispecific polypeptide construct of embodiment 112, whereinthe agent is conjugated to the multispecific polypeptide construct via alinker.

114. A polynucleotide(s) encoding the mutispecific polypeptideconstructs of any of embodiments 1-113.

115. A polynucleotide encoding a polypeptide chain of any of themutispecific polypeptide constructs of any of embodiments 1-113.

116. A polynucleotide, comprising a first nucleic acid sequence encodinga first polypeptide of a mutispecific construct of any of embodiments1-115 and a second nucleic acid sequence encoding a second polypeptideof the mutispecific construct, wherein the first and second nucleic acidsequence are separated by an internal ribosome entry site (IRES), or anucleic acid encoding a self-cleaving peptide or a peptide that causesribosome skipping.

117. The polynucleotide of embodiment 116, wherein the first nucleicacid sequence and second nucleic acid sequence are operably linked tothe same promoter.

118. The polynucleotide of embodiment 116 or embodiment 117, wherein themultispecific polypeptide construct comprises a third polypeptide chain,and the polynucleotide further comprises a third nucleic acid encodingthe third polypeptide of the mutispecific construct.

119. The polynucleotide of embodiment 118, wherein the third nucleicacid is separated from the first and/or second polypeptide by aninternal ribosome entry site (IRES), or a nucleic acid encoding aself-cleaving peptide or a peptide that causes ribosome skipping and/orthe third nucleic acid sequence is operably linked to the same promoteras the first and/or second nucleic acid sequence.

120. The polynucleotide of any of embodiments 116-119, wherein thenucleic acid encoding a self-cleaving peptide or a peptide that causesribosome skipping is selected from a T2A, a P2A, a E2A or a F2A.

121. A vector, comprising the polynucleotide of any of embodiments114-120.

122. The vector of embodiment 121 that is an expression vector.

123. The vector of embodiment 121 or 122 that is a viral vector or aeukaryotic vector, optionally wherein the eukaryotic vector is amammalian vector.

124. A cell, comprising polynucleotide or polynucleotides of any ofembodiments 114-120, or a vector or vectors of any of embodiments121-123.

125. The cell of embodiment 124, wherein the cell is recombinant orisolated.

126. The cell of embodiment 125, wherein the cell is a mammalian cell.

127. The cell of embodiment 126, wherein the cell is a HEK293 or CHOcell.

128. A method of producing a multispecific polypeptide construct, themethod comprising introducing into a cell a polynucleotide orpolynucleotides of any of embodiments 114-120 or a vector or vectors ofany of embodiments 121-123 and culturing the cell under conditions toproduce the multispecific polypeptide construct.

129. A method of producing a multispecific polypeptide construct, themethod comprising culturing the cell of any of embodiments 124-127 underconditions in which the multispecific polypeptide is produced by thecell.

130. The cell of embodiment 128 or 129, wherein the cell is a mammaliancell.

131. The cell of embodiment 130, wherein the cell is a HEK293 or CHOcell.

132. The method of embodiment 128 or embodiment 129, further comprisingisolating or purifying the multispecific polypeptide construct from thecell.

133. The method of any of embodiments 128-132, wherein the multispecificpolypeptide construct is a heterodimer.

134. A multispecific polypeptide construct produced by the method of anyof embodiments 128-133.

135. A pharmaceutical composition comprising the multispecificpolypeptide construct of any of embodiments 1-113 or embodiment 134 anda pharmaceutically acceptable carrier.

136. The pharmaceutical composition of embodiment 135 that is sterile.

137. A method of stimulating or inducing an immune response, the methodcomprising contacting a target cell and a T cell with the multispecificpolypeptide construct of any of embodiments 1-113 or embodiment 134 orthe pharmaceutical composition of embodiments 109 or embodiment 110,said target cell expressing a tumor associated antigen recognized by themultispecific polypeptide construct.

138. The method of embodiment 137, wherein the target cell is a tumorcell expressing the tumor associated antigen (TAA).

139. The method of embodiment 137 or embodiment 138, wherein thecontacting is carried out ex vivo or in vitro.

140. The method of any of embodiments 137-149, wherein the contacting iscarried out in vivo in a subject.

141. A method of stimulating or inducing an immune response in asubject, the method comprising administering, to a subject in needthereof, a therapeutically effective amount of the multispecificconjugate of any of embodiments 1-113 or embodiment 134 or thepharmaceutical composition of embodiments 109 or embodiment 110.

142. The method of any of embodiments 137-141, which increasescell-mediated immunity.

143. The method of any of embodiments 137-142, which increases T-cellactivity.

144. The method of any of embodiments 137-143, which increases cytolyticT-cell (CTL) activity.

145. The method of any of embodiments 137-144, wherein the immuneresponse is increased against a tumor or cancer.

146. The method of any of embodiments 137-145, wherein the method treatsa disease or condition in the subject.

147. A method of treating a disease or condition in a subject, themethod comprising administering, to a subject in need thereof, atherapeutically effective amount of the multispecific conjugate of anyof embodiments 1-113 or the pharmaceutical composition of embodiments135 or embodiment 136.

148. The method of embodiment 146 or embodiment 147, wherein the diseaseor condition is a tumor or a cancer.

149. The method of any of embodiments 140-148, wherein said subject is ahuman.

VI. EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1: Method of Producing Multispecific Constructs With ConstrainedCD3 Binding

Example 1 describes the generation and expression of multispecificpolypeptide constructs containing a CD3 binding region that exhibitsconstrained CD3 binding. The multispecific constructs were generated invarious configurations, as shown in FIG. 1, FIGS. 2A-2B, FIGS. 3A-3C andFIGS. 4A-4B, to contain a heterodimeric Fc region of an immunoglobulincoupled by a linker (e.g. a non-cleavable linker) to the CD3 bindingregion, and one or more antigen binding domains that binds a tumorassociated antigen (TAA) positioned amino-terminally relative to the Fcregion and/or carboxy-terminally relative to the CD3 binding region ofthe multispecific polypeptide construct.

A. Design and Generation of Constructs

Polynucleotides encoding at least a first polypeptide chain and a secondpolypeptide chain of the heterodimeric multispecific polypeptideconstruct were generated and cloned into a plasmid for expression. Thefirst polypeptide chain generally included in order, from the N-terminusto C-terminus, a first Fc polypeptide (e.g. an Fc hole polypeptide); anon-cleavable linker; and a variable light (VL) domain of an anti-CD3antibody. The second polypeptide chain generally included in order, fromthe N-terminus to C-terminus, a second Fc polypeptide (e.g. an Fc knobpolypeptide); the same non-cleavable linker as the first polypeptidechain; and a variable heavy (VH) domain of an anti-CD3 antibody. Theanti-CD3 antibody included either a disulfide-stabilized (dsFv) antibody(anti-CD3 VH with the mutation G44C and VL with the mutation G100C) orcontained a non-disulfide stabilized Fv antibody, as set forth in TablesE1.1 and E1.2. Various exemplary Fc polypeptide pairs to facilitateheterodimerization of the polypeptide chains were used as set forth inTables E1.1 and Table E1.2. One or both of the polypeptide chainsadditionally encoded one or more TAA antigen binding domainamino-terminal to the Fc domain and/or carboxy-terminal to the CD3binding region, in various configurations. Similar constructs can begenerated using other heterodimeric Fc configurations, including otherknob-into-hole configurations, such as any as described; otherCD3-binding regions, including other anti-CD3 antibodies, including dsFvor other monovalent fragments; or other TAA antigen-binding fragments,such as scFv, sdAb or Fab formats can also be used.

Among generated constructs, the non-cleavable linker included linkersranging from 3-18 amino acids in size. Examples of non-cleavable linkersused in exemplary generated molecules were GGS (e.g. contained inexemplary construct cx1356), GGSGGS (SEQ ID NO:10, contained inexemplary construct cx1357), GGSGGSGGS (SEQ ID NO:11, contained inexemplary construct cx1358), GGSGGSGGSGGS (SEQ ID NO:12, contained inexemplary construct cx1359), GGSGGSGGSGGSGGS (SEQ ID NO:13, contained inexemplary construct cx1360), and GGGGGSGGGGGSGGGGGS (SEQ ID NO:119,contained in exemplary construct cx5823 and cx5952) orGGSGGGGSGGGGSGGGGS (SEQ ID NO: 147, contained in exemplary constructcx681).

Any antigen binding domain that binds to a TAA can be employed in theprovided multispecific polypeptide constructs. Exemplary generatedproteins contained an antigen binding domain that binds Folate ReceptorAlpha (FRα), B7H3 (CD276), or Delta-like 3 (DLL3). The antigen-bindingdomain can include single chain fragments (e.g. sdAb or scFv) or twochain antigen-binding fragments (Fabs). When the TAA was provided as asingle chain fragment, e.g. sdAb or scFv, the TAA antigen binding domainwas linked at the N-terminus to one or both polypeptide chains of the Fcheterodimer (e.g. hole and/or knob) by a peptide linker, e.g. PGGGG (SEQID NO:102) and/or was linked at the C-terminus to one or both domains(e.g. VH and/or VL) of the CD3 binding region by a peptide linker, e.g.GGGG (SEQ ID NO:103). Other similar peptide linkers can be employed.When the TAA was provided as a Fab antigen-binding fragment theconstruct was composed of a VH and CH1 linked directly to one or both Fcpolypeptides without a linker, as well as a light chain composed of a VLand CL. These TAA binding Fabs can be located on the amino- orcarboxy-terminus of the heterodimeric Fc.

Multispecific polypeptide constructs were generated containing 1, 2, 3or 4 TAA antigen binding domain, such as to provide for monovalent,bivalent, trivalent, or tetravalent binding, respectively. In somecases, the TAA antigen binding domains were the same (mono-epitopic). Insome cases, the TAA antigen binding domains were different, such thatthe generated multispecific polypeptide constructs exhibited specificityfor at least two different TAAs, to different epitopes of the same TAA(bi-epitopic) or the same epitopes of the same TAA (mono-epitopic).

Among the generated proteins were constructs in which the TAA antigenbinding domains were composed as single domain antibodies (sdAbs).Polynucleotides were generated to encode polypeptide chains of exemplarymultispecific polypeptide constructs containing non-cleavable linkers.These included constructs designated cx1356, cx1357, cx1358, cx1359,cx1360, and cx681, targeting FRα as depicted in FIG. 2B; cx3072, cx5952,cx6079, cx6080, cx6081, cx5823, cx5873, and cx5965, targeting B7H3 asdepicted in FIGS. 3A and 3B; and cx5352, cx5499, cx5800, and cx5801targeting DLL3 as depicted in FIGS. 4A and 4B. Notably, some constructswere generated wherein the VH domain of the dsFv anti-CD3 antibody andthe sdAb were both linked to the same side (e.g. hole or knob side) ofthe Fc heterodimer (e.g. cx3072 and cx5952, shown in FIG. 3A).Constructs were engineered without a disulfide stabilized Fv or wereengineered with a disulfide linkage stabilizing the VH and VL domains ofthe anti-CD3 antibody. Some of the exemplary constructs generatedadditionally contained a sdAb (containing a CDR1, a CDR2 and a CDR3 setforth in SEQ ID Nos: 221, 222 and 223, respectively) targeting 4-1BBco-stimulatory receptor (e.g. cx5823, cx5873, cx5965, cx5352, cx5801,cx5800). A list of exemplary constrained CD3 binding constructs havingsdAb TAA domains is given below in Table E1.1.

TABLE E1.1 Exemplary Constrained CD3 engaging constructs N-termConstruct sdAb CD3 Binding C-term sdAb Disulfide ID Chain (Target) FcLinker Domain (Target) Stabilized cx681 1 FRα IgG1-Knob GGSGGGGSGGGGSVH34 (SEQ ID FRα sdAb2 yes sdAb1 (SEQ ID GGGGS (SEQ ID NO: 198) (SEQ ID(SEQ ID NO: 82, 86 NO: 147) NO: 121) NO: 120) or 201) 2 FRα IgG1-HoleGGSGGGGSGGGGS VL21 (SEQ ID FRα sdAb2 sdAb1 (SEQ ID GGGGS (SEQ IDNO: 200) (SEQ ID (SEQ ID NO: 83, 87 NO: 147) NO: 121) NO: 120) or 202)cx1356 1 FRα IgG1-Knob GGS VH34 (SEQ ID FRα sdAb2 yes sdAb1 (SEQ IDNO: 198) (SEQ ID (SEQ ID NO: 82, 86 NO: 121) NO: 120) or 201) 2 FRαIgG1-Hole GGS VL21 (SEQ ID FRα sdAb2 sdAb1 (SEQ ID NO: 200) (SEQ ID(SEQ ID NO: 83, 87 NO: 121) NO: 120) or 202) cx1357 1 FRα IgG1-KnobGGSGGS (SEQ ID VH34 (SEQ ID FRα sdAb2 yes sdAb1 (SEQ ID NO: 10) NO: 198)(SEQ ID (SEQ ID NO: 82, 86 NO: 121) NO: 120) or 201) 2 FRα IgG1-HoleGGSGGS (SEQ ID VL21 (SEQ ID FRα sdAb2 sdAb1 (SEQ ID NO: 10) NO: 200)(SEQ ID (SEQ ID NO: 83, 87 NO: 121) NO: 120) or 202) cx1358 1 FRαIgG1-Knob GGSGGSGGS (SEQ VH34 (SEQ ID FRα sdAb2 yes sdAb1 (SEQ IDID NO: 11) NO: 198) (SEQ ID (SEQ ID NO: 82, 86 NO: 121) NO: 120) or 201)2 FRα IgG1-Hole GGSGGSGGS (SEQ VL21 (SEQ ID FRα sdAb2 sdAb1 (SEQ IDID NO: 11) NO: 200) (SEQ ID (SEQ ID NO: 83, 87 NO: 121) NO: 120) or 202)cx1359 1 FRα IgG1-Knob GGSGGSGGSGGS  VH34 (SEQ ID FRα sdAb2 yes sdAb1(SEQ ID (SEQ ID NO: 12) NO: 198) (SEQ ID (SEQ ID NO: 82, 86 NO: 121)NO: 120) or 201) 2 FRα IgG1-Hole GGSGGSGGSGGS VL21 (SEQ ID FRα sdAb2sdAb1 (SEQ ID (SEQ ID NO: 12) NO: 200) (SEQ ID (SEQ ID NO: 83, 87NO: 121) NO: 120) or 202) cx1360 1 FRα IgG1-Knob GGSGGSGGSGGSGVH34 (SEQ ID FRα sdAb2 yes sdAb1 (SEQ ID GS NO: 198) (SEQ ID (SEQ IDNO: 82, 86 (SEQ ID NO: 13) NO: 121) NO: 120) or 201) 2 FRα IgG1-HoleGGSGGSGGSGGSG VL21 (SEQ ID FRα sdAb2 sdAb1 (SEQ ID GS NO: 200) (SEQ ID(SEQ ID NO: 83, 87 (SEQ ID NO: 13) NO: 121) NO: 120) or 202) cx5823 1B7H3 xELL- GGGGGSGGGGGSG VH13 (SEQ ID B7H3 sdAb5 yes sdAb4 Knob (SEQGGGGS (SEQ ID NO: 44) (SEQ ID (SEQ ID ID NO: 88) NO: 119) NO: 216)NO: 214) 2 None xELL-Hole GGGGGSGGGGGSG VL10 (SEQ ID Co-stim (SEQ IDGGGGS (SEQ ID NO: 72) Receptor NO: 93) NO: 119) sdAb cx5952 1 B7H3 xELL-GGGGGSGGGGGSG VH13 (SEQ ID B7H3 sdAb5 yes sdAb4 Knob (SEQ GGGGS (SEQ IDNO: 44) (SEQ ID NO: (SEQ ID ID NO: 88) NO: 119) 216) NO: 214) 2 NonexELL-Hole GGGGGSGGGGGSG VL10 (SEQ ID None (SEQ ID GGGGS (SEQ ID NO: 72)NO: 93) NO: 119) cx6079 1 B7H3 Fc-Het-1 GGGGSGGGGSGGG VH32 (SEQ ID Noneno sdAb4 (SEQ ID GS NO: 196) (SEQ ID NO: 194) (SEQ ID NO: 170) NO: 214)2 B7H3 Fc-Het-2 GGGGSGGGGSGGG VL20 (SEQ ID None sdAb4 (SEQ ID GSNO: 199) (SEQ ID NO: 195) (SEQ ID NO: 170) NO: 214) cx6080 1 B7H3Fc-Het-1 GGGGSGGGGSGGG VH33 (SEQ ID None yes sdAb4 (SEQ ID GS NO: 197)(SEQ ID NO: 194) (SEQ ID NO: 170) NO: 214) 2 B7H3 Fc-Het-2 GGGGSGGGGSGGGVL21 (SEQ ID None sdAb4 (SEQ ID GS  NO: 200) (SEQ ID NO: 195)(SEQ ID NO: 170) NO: 214) cx6081 1 B7H3 Fc-Het-1 GGGGSGGGGSGGGVH13 (SEQ ID None yes sdAb4 (SEQ ID GS NO: 44) (SEQ ID NO: 194)(SEQ ID NO: 170) NO: 214) 2 B7H3 Fc-Het-2 GGGGSGGGGSGGG VL10 (SEQ IDNone sdAb4 (SEQ ID GS NO: 72) (SEQ ID NO: 195) (SEQ ID NO: 170) NO: 214)cx5841 1 B7H3 xELL- GGGGGSGGGGGSG VH13 (SEQ ID B7H3 sdAb3 yes sdAb4Knob (SEQ GGGGS (SEQ ID NO: 44) (SEQ ID (SEQ ID ID NO: 84 NO: 119)NO: 217) NO: 214) 88 or 203) 2 None xELL-Hole GGGGGSGGGGGSG VL10 (SEQ IDCo-stim (SEQ ID GGGGS (SEQ ID NO: 72) Receptor NO: 91, 93 NO: 119) sdAbor 206) cx5187 1 B7H3 xELL- GGGGGSGGGGGSG VH13 (SEQ ID B7H3 sdAb3 yessdAb4 Knob (SEQ GGGGS (SEQ ID NO: 44) (SEQ ID (SEQ ID ID NO: 84,NO: 119) NO: 217) NO: 214) 88 or 203) 2 None xELL-Hole GGGGGSGGGGGSGVL10 (SEQ ID Co-stim (SEQ ID GGGGS (SEQ ID NO: 72) Receptor NO: 91, 93NO: 119) sdAb or 206) cx5499 1 DLL3 xELL- GGGGGSGGGGGSG VH13 (SEQ IDDLL3 sdAb2 yes sdAb1 Knob (SEQ GGGGS (SEQ ID NO: 44) (SEQ ID NO: (SEQ IDID NO: 84, NO: 119) 220) NO: 219) 88 or 203) 2 None xELL-HoleGGGGGSGGGGGSG VL10 (SEQ ID None (SEQ ID GGGGS (SEQ ID NO: 72) NO: 91, 93NO: 119) or 206) cx3072 1 B7H3 IgG1-Knob GGGGGSGGGGGSG VH13 (SEQ IDB7H3 sdAb1 yes sdAb2 (SEQ ID GGGGS (SEQ ID NO: 44) (SEQ ID (SEQ IDNO: 82, 86 NO: 119) NO: 218) NO: 215) or 201) 2 None IgG1-KnobGGGGGSGGGGGSG VL10 (SEQ ID None (SEQ ID GGGGS (SEQ ID NO: 72) NO: 83, 87NO: 119) or 202) cx5873 1 None xELL- GGGGGSGGGGGSG VH13 (SEQ IDB7H3 sdAb3 yes Knob (SEQ GGGGS (SEQ ID NO: 44) (SEQ ID ID NO: 84,NO: 119) NO: 217) 88 or 203) 2 None xELL-Hole GGGGGSGGGGGSG VL10 (SEQ IDCo-stim (SEQ ID GGGGS (SEQ ID NO: 72) Receptor NO: 91, 93 NO: 119) sdAbor 206) cx5965 1 B7H3 xELL- GGGGGSGGGGGSG VH13 (SEQ ID none yes sdAb4Knob (SEQ GGGGS (SEQ ID NO: 44) (SEQ ID ID NO: 84, NO: 119) NO: 214)88 or 203) 2 None xELL-Hole GGGGGSGGGGGSG VL10 (SEQ ID Co-stim (SEQ IDGGGGS (SEQ ID NO: 72) Receptor NO: 91, 93 NO: 119) sdAb or 206) cx5352 1DLL3 xELL- GGGGGSGGGGGSG VH13 (SEQ ID DLL3 sdAb2 yes sdAb1 Knob (SEQGGGGS (SEQ ID NO: 44) (SEQ ID NO: (SEQ ID ID NO: 84, NO: 119) 220)NO: 219) 88 or 203) 2 None xELL-Hole GGGGGSGGGGGSG VL10 (SEQ ID Co-stim(SEQ ID GGGGS (SEQ ID NO: 72) Receptor NO: 91, 93 NO: 119) sdAb or 206)cx5800 1 DLL3 xELL- GGGGGSGGGGGSG VH13 (SEQ ID None yes sdAb1 Knob (SEQGGGGS (SEQ ID NO: 44) (SEQ ID ID NO: 84, NO: 119) NO: 219) 88 or 203) 2None xELL-Hole GGGGGSGGGGGSG VL10 (SEQ ID Co-stim (SEQ ID GGGGS (SEQ IDNO: 72) Receptor NO: 91, 93 NO: 119) sdAb or 206) cx5801 1 None xELL-GGGGGSGGGGGSG VH13 (SEQ ID DLL3 sdAb2 yes Knob (SEQ GGGGS (SEQ IDNO: 44) (SEQ ID NO: ID NO: 84, NO: 119) 220) 88 or 203) 2 None xELL-HoleGGGGGSGGGGGSG VL10 (SEQ ID Co-stint (SEQ ID GGGGS (SEQ ID NO: 72)Receptor NO: 91, 93 NO: 119) sdAb or 206)

Exemplary generated constructs also included constructs in which the TAAantigen binding domains were composed as a Fab (designated MAB formats).In this example, an anti-B7H3 Fab was used composed of a heavy chain Fdset forth in SEQ ID NO:127 and a light chain set forth in SEQ ID NO:128.Polynucleotides were generated to encode polypeptide chains of exemplarymultispecific polypeptide constructs containing non-cleavable linkers.These included cx5067, cx6083, and cx6084, as depicted in FIG. 3C.Constructs were engineered without a disulfide stabilized anti-CD3antibody Fv or were engineered with a disulfide linkage stabilizing theVH and VL domains of the anti-CD3 antibody (anti-CD3 VH with themutation G44C and VL with the mutation G100C). A list of exemplary Fabconstructs is provided below in Table E1.2.

TABLE E1.2 Exemplary B7H3-targeted constrained CD3 engaging constructsConstruct N-term CD3 Binding C-term sdAb Disulfide ID Chain Domain FcLinker Domain (Target) Stabilized cx5067 1 B7H3-Fab Fc-Het-1GGGGSGGGGSGGGG VH32 (SEQ ID None no (SEQ ID (SEQ ID S NO: 196) NOs: 127,NO: 194) (SEQ ID NO: 170) 128) 2 B7H3-Fab Fc-Het-2 GGGGSGGGGSGGGGVL20 (SEQ ID None (SEQ ID (SEQ ID S NO: 199) NOs: 127, NO: 195)(SEQ ID NO: 170) 128) cx6083 1 B7H3-Fab Fc-Het-1 GGGGSGGGGSGGGGVH33 (SEQ ID None yes (SEQ ID (SEQ ID S NO: 197) NOs: 127, NO: 194)(SEQ ID NO: 170) 128) 2 B7H3-Fab Fc-Het-2 GGGGSGGGGSGGGG VL21 (SEQ IDNone (SEQ ID (SEQ ID S NO: 200) NOs: 127, NO: 195) (SEQ ID NO: 170) 128)cx6084 1 B7H3-Fab Fc-Het-1 GGGGSGGGGSGGGG VH13 (SEQ ID None yes (SEQ ID(SEQ ID S NO: 44) NOs: 127, NO: 194) (SEQ ID NO: 170) 128) 2 B7H3-FabFc-Het-2 GGGGSGGGGSGGGG VL10 (SEQ ID None (SEQ ID (SEQ IDS (SEQ ID NO: 170) NO: 72) NOs: 127, NO: 195) 128)

B. Expression and Purification of Generated Constructs

Separate plasmids encoding each chain of the heterodimeric constrainedCD3 binding protein were transiently transfected at an equimolar ratiointo mammalian cells (either HEK293 or CHO) using polyethylenimine.Recombinant protein secreted into the supernatant was collected after3-14 days, and purified by protein A chromatography, followed by eitherpreparative size exclusion chromatography (SEC) or flow-throughhydrophobic interaction chromatography (HIC). In some cases,heterodimeric protein was enriched for during purification due to amutation designed into one chain of the heterodimeric Fc at positionI253R or H435R (e.g. in the hole-Fc) such that it did not bind proteinA, and thus homodimers of I253R or H435R were not purified. The secondchromatography step by SEC (AKTA with Superdex-200 resin) or FT-HIC(AKTA with butyl/phenyl sepharose) was used to remove undesiredcross-paired species containing two heterodimeric Fcs that were morehydrophobic and twice the expected molecular weight.

The method favored production of heterodimeric multispecific polypeptideconstructs, containing properly paired species of heterodimeric Fc andthe anti-CD3 Fv (e.g. disulfide stabilized anti-CD3 Fv). Purifiedheterodimeric constrained CD3 binding protein was stable and did notaccumulate cross-paired species upon prolonged incubation at 4° C. orincreased protein concentration.

Example 2: Assessment of Binding of Constrained CD3 Binding Constructsto Cancer Cells and Primary T Cells by Flow Cytometry

This Example describes studies assessing binding of exemplary constructsto T cells or to cancer cells. These studies were carried out in singlecultures containing either only the T cells or only the cancer cells inisolation from each other.

A. FR-Alpha Binding

Binding of exemplary multispecific polypeptide constructs of thedisclosure containing an antigen-binding domain directed against FolateReceptor Alpha (FRα) to CD3 on the surface of primary T cells and to FRαexpressing cells (Ovcar-5) was assessed by flow cytometry. The tumorantigen binding domains of the tested constructs bind the FolateReceptor Alpha (FRα), which is not expressed on the primary T cells. Thetested constructs included cx1356 and cx681, containing a non-cleavablelinker of 3 amino acids in cx1356 or a non-cleavable linker of 18 aminoacids in cx681 (see FIG. 2B and Table E1.1).

For the studies described in FIGS. 5A and 5B, 100 nM of each construct,cx1356 or cx681 was used and assessed for binding to either the Ovcar-5cells or primary T-cells. An anti-CD3 antibody was included as acontrol. A titration of the constrained CD3 engaging constructs oneither FRα expressing cells (Ovcar-5; FIG. 5C) or isolated primaryT-cells (FRα negative; FIG. 5D), was conducted to assess binding. Boundconstructs were detected using fluorophore-conjugated anti-human IgG Fcsecondary antibody.

The tested FRα-targeting constructs with various linkers between the Fcand the component of the CD3 binding domains were found to bind FRαexpressing cells (Ovcar-5) (FIGS. 5A and 5C), but lacked the capacity tobind T-cells (FIGS. 5B and 5D). These results are consistent with afinding that binding to CD3 on T cells in isolation is constrained inthe provided formats.

B. B7H3 Binding

Binding of exemplary multispecific constructs containing anantigen-binding domain directed against B7H3 were assessed for bindingto B7H3 positive A375 tumor cells or primary T-cells. The constructswere generated containing antigen-binding domain(s) that were eithersdAbs or a FABs, and that were positioned either only N-terminal to theFc or both N-terminal to the Fc and C-terminal to the anti-CD3 bindingdomain (see FIGS. 3A and 3C and Table E1.1). Among the various formatsof constructs tested included: sdAb-Fc-dsFV-sdAb (cx3072, cx5952)sdAb-Fc-FV (cx6079), sdAb-Fc-dsFV (c56080, cx6081), MAB-FV (cx5067) andMAB-dsFV (cx6083, cx6084), where the FV represents the anti-CD3 bindingdomain composed of VH and VL domain pairs and “ds” notes disulfidestabilized via an engineered interdomain disulfide bond.

FIGS. 6A-F demonstrate that these constructs are capable of binding toB7H3 but not T-cells in isolation. Binding was assessed as describedabove via flow cytometry using a fluorophore-conjugated anti-human Fcsecondary antibody. cx3072 bound to A375 cells with high affinity (FIG.6A) but not to isolated T-cells (FIG. 6B). The tested sdAb-Fc-dsFV-sdAb(cx5952) displayed higher binding affinity compared to a FAB containingMAB-FV (cx5067) and MAB-dsFV constructs (cx6083, cx6084) (FIG. 6C). TheB7H3-targeting sdAb containing constructs (cx5952, cx6079, cx6080, andcx6081) bound to B7H3 positive cells with similar affinities (depictedin FIG. 6E), with cx5952 displaying higher maximal binding. cx6079,cx6080 and cx6081 contain two identical B7H3-targeting sdAbs, whereascx5952, and cx3072 contain two distinct B7H3-targeting sdAb that binddifferent epitopes. The MAB-FV, cx5067, contains two identicalB7H3-targeting FAB domains. Notably none of the exemplary B7H3-targetedconstrained CD3 engaging constructs bound isolated primary humanT-cells, as depicted in FIGS. 6B, 6D, and 6F. These results furthersupport that binding to CD3 on T cells in isolation is constrained inthe provided formats.

Example 3: Assessment of CD3 Signaling Activity in Co-Cultures withAntigen-Expressing Target Cells and Impact of Linker Length on Activity

The effect of various length linkers between the Fc and the componentdomains (VH and VL) that comprise the CD3 binding region on T-cellactivating capacity was tested using a Jurkat reporter assay. The CD3reporter cells were developed from Jurkat cells that naturally expressCD3 and were engineered to express NFAT-driven green fluorescenceprotein (GFP). Agonism of CD3 results in NFAT signaling and productionof green fluorescence.

Antigen targeting constrained CD3 engaging constructs were titrated ontoco-cultures of target cells and Jurkat CD3 reporter cells. In thisassay, target cells included either IGROV1 (FRα positive) or NCI-460(FRα negative). For reporter assays utilizing adherent antigenexpressing target cells, target cells were seeded, allowed to settle atroom temperature for uniform distribution, and incubated for severalhours at 37° C. to permit adherence prior to addition of reporter cellsand antigen targeting constrained CD3 engaging constructs. Assay plateswere serially imaged using an IncuCyte ZOOM system and CD3 reporter cellactivation was determined by measuring GFP expression as the totalintegrated green object in the well.

FRα-targeting constrained CD3 engaging constructs, generated asdescribed in Example 1 containing GlySer-based linkers of varyinglengths as listed in Table E3 were used in these assays.

TABLE E3 Tested Linker Lengths SEQ ID NO Linker — gs3: GGS  10gs6: GGSGGS  11 gs9: GGSGGSGGS  12 gs12: GGSGGSGGSGGS  13gs15: GGSGGSGGSGGSGGS 119 gs18: GGGGGSGGGGGSGGGGGS 147gs18: GGSGGGGSGGGGSGGGGS

As shown in FIGS. 7A-7F, the T-cell activating activity as determined byfluorescent intensity was dependent on co-culture with FRα-antigenexpressing target cells. The length of the linker and T-cell activatingcapacity were positively correlated. T-cell activating capacity wasshown to directly relate to linker length, indicating shorter linkersrestrict CD3 binding to a greater extent (see FIGS. 7A, 7C and 7E).Importantly, T-cell engagement of the constructs is dependent onTAA-binding, as these constructs did not demonstrate a T-cell bindingcapacity in isolation (e.g. solution form when unbound to target TAA) asshown above in Example 2. Further, no observable fluorescence wasmeasured in co-cultures with FRα negative cells (FIGS. 7B, 7D and 7E).Together, these constructs displayed restricted or substantially reducedbinding to CD3, yet were capable of activating T-cells in a targetdependent manner.

Example 4: Assessment of T Cell Activating Activity of B7I13-TargetedConstrained CD3 Binding Constructs

Constructs containing either a B7H3-targeted sdAb or a Fab as thetumor-associated antigen-binding domain were assessed for T-cellactivating activity in a T cell reporter assay and in a T cellcytotoxicity assay. Activity of B7H3-targeted constrained CD3 engagingconstructs that were formatted with an anti-B7H3 sdAb (e.g. cx5823,cx6079, cx6080, cs6081, cx3072 and cx5952) or anti-B7H3 MAB constructsformatted with a Fab (e.g. cx5067, cx6083 or cx6084) as theantigen-binding domain(s) were assessed (see FIGS. 3A-3C and TableE1.1). All tested constructs, except cx5067 and cx6079, contained adisulfide-stabilized anti-CD3 Fv (dsFv) containing an interchaindisulfide bond created by the modification of anti-CD3 VH G44C pairedwith VL G100C. The anti-CD3 Fv of cx5067, designated MAB-Fv, was notdisulfide-stabilized.

A. T cell Reporter Activity

The NFAT-GFP CD3 Jurkat reporter described in Example 3 was used tocompare the CD3 agonistic properties of B7H3-targeted constrained CD3engaging constructs when co-cultured in the presence of B7H3-positivecells (A375) or non-target CCRF-CEM cells that naturally lack B7H3expression. In this assay, anti-B7H3 sdAb constructs, cx5823, cx6079,cx6080 and cx6081, or anti-B7H3 Fabs constructs, cx5067, cx6083 andcx6084, were used as the B7H3-targeting domains. As shown in FIG. 8A,the constructs containing B7H3-targeted sdAb displayed similar potenciesof antigen-dependent CD3 activation. As shown in FIG. 8C the exemplarycx5823 construct containing B7H3-targeted sdAbs was found to be superiorat mediating antigen-dependent CD3 activation compared to the constructscontaining B7H3-targeted Fabs. Although cx5823 is formatted with abinding domain for a costimulatory receptor, it is unlikely that thiscomponent contributed to the difference in results, since Jurkat T cellsdo not express the costimulatory receptor. None of the constructsdemonstrated activity against the B7H3-negative CCRF-CEM cells (FIGS. 8Band 8D).

B. Cytotoxicity

To further assess activity of the molecules, exemplary B7H3-targetedconstructs cx3072 and cx5952 (each formatted as sdAb-dsFv), cx6083 andcx6084 (MAB-dsFv), cx5067 (MAB-Fv), cx6079 (sdAb-Fv), and cx6080 and cx6081 (sdAb-dsFv) were tested in a T-cell-mediated cytotoxicity assay.Target cells included the B7H3 positive cell line, A375, and eithermodified A375 cells, wherein B7H3 gene was disrupted by CRISPR(A375:B7H3 KD), or CCRF-CEM cells that naturally lacked B7H3 expression.Target cells were seeded at 1.0×10⁴ cells per well, allowed to settle atroom temperature for uniform distribution, and incubated for severalhours at 37° C. Primary T cells were negatively enriched from PBMCsisolated from healthy human donor leukopaks and added at a 10:1 Tcell-to-target cell ratio. Green caspase-3/7 reagent was added, whichfluorescently labels nuclear DNA of cells undergoing apoptosis wasadded. Multispecific constructs with constrained CD3 engaging activitywere titrated onto the co-culture and assay plates were serially imagedusing an IncuCyte ZOOM system. Target cell death was determined bymeasuring total red/green overlap object area.

As shown in FIGS. 9A and 9B, exemplary constructs cx3072 and cx5952containing sdAb B7H3-targeted antigen-binding domains induced potentT-cell-mediated cytotoxicity of B7H3 positive (A375) but not B7H3negative cell lines.

When compared to exemplary B7H3-targeting constrained CD3 engagers withFab B7H3-targeting domains (cx5067, cx6083 and cx6084), the exemplarycx5952 sdAb B7H3-targeting constrained CD3 engager mediated enhancedtarget-dependent T-cell cytotoxicity (FIG. 10A). No measurable T cellcytotoxicity was observed against the B7H3 negative cell line CCRF-CEMfor any of the tested constructs, consistent with the capacity topotently induce antigen-dependent T-cell activation (FIG. 10B). Of theconstructs tested, representative MAB-dsFV constructs cx6084 and cx6083contained the engineered disulfide, whereas the representative MAB-FVconstruct cx5067 lacked this stabilizing modification. Notably cx6083and cx5067 are identical with the exception of the presence (cx6083) orabsence (cx5067) of the engineered disulfide within the anti-CD3 FVdomain (depicted in FIG. 3C). The engineered disulfide was created bythe modification of G44C within VH and G100C within VL. As shown in FIG.10A, cx6083 displayed superior potency in mediating target-dependentT-cell cytotoxicity compared to cx5067, suggesting that theincorporation of the inter-domain disulfide is beneficial in T-cellmediated cytotoxicity, likely by enhancing proper association of the VHand VL domains that comprise the anti-CD3 FV.

When compared to other exemplary B7H3-targeting constrained CD3 engagerswith sdAb B7H3-targeting domains (cx6079, cx6080 and cx6081), theexemplary cx5952 sdAb B7H3-targeting constrained CD3 engager mediatedenhanced target-dependent T-cell cytotoxicity (FIG. 10C). No measurableT cell cytotoxicity was observed against the B7H3 negative cell lineCCRF-CEM for any of the tested constructs, consistent with the capacityto potently induce antigen-dependent T-cell activation (FIG. 10D). Ofthe constructs tested, sdAb-dsFV constructs cx5952, cx6080 and cx6081contained the engineered disulfide linkage, whereas the sdAb-FVconstruct cx6079 lacked this stabilizing modification. The engineereddisulfide was created by the modification of G44C within VH and G100Cwithin VL. Notably, cx5952 was engineered to have two distinct B7H3targeting domains, one located at the amino terminal and one located atthe carboxy terminal. cx6079, cx6080, and cx6081 were engineered to havetwo identical B7H3 targeting domains, both located at the amino terminal(see FIG. 3A).

C. T Cell Modulation

To further assess T cell modulation, exemplary multispecific CD3constrained binding constructs was assessed by monitoring the ability ofthe constructs to modulate T cell activation markers. To assess T cellactivation, suspension cells from T cell cytotoxicity assays above,involving culture of T cells with B7H3 positive (A375) or B7H3 negativecell lines (CCRF-CEM) in the presence of an exemplary B7H3-targetedconstrained CD3 engaging construct, cx5952, were collected. Cells werestained with a live/dead stain and fluorophore-conjugated anti-CD4,anti-CD8, anti-CD25, anti-CD69, and/or anti-CD71 antibodies. Cells wereanalyzed using a SONY SA3800 spectral analyzer and CD4+ or CD8+ T cellactivation was determined by measuring expression levels of CD25, CD69or CD71 or percent CD25-, CD69- or CD71-positive.

Results are shown for CD25 expression (FIG. 11A), CD69 expression (FIG.11B) and CD71 expression (FIG. 11C) on CD4+ and CD8+ T cells followingthe co-culture with B7H3 positive (A375) or B7H3 negative cell lines(CCRF-CEM) in the presence of cx5952. The results showed that cx5952mediated a dose-dependent B7H3-dependent T-cell activation via CD3binding, as evidenced by increased expression of CD25, CD69 and CD71 inCD4+ and CD8+ T cells.

When compared to the other exemplary B7H3-targeting constrained CD3engagers with sdAb B7H3-targeting domains (cx6079, cx6080 and cx6081),the exemplary cx5952 sdAb B7H3-targeting constrained CD3 engagermediated increased T cell activation as evidenced by increasedexpression of CD25 in CD4+ T cells (FIG. 11D) and in CD8+ T cells (FIG.1111) and increased expression of CD71 in CD4+ T cells (FIG. 11F) and inCD8+ T cells (FIG. 11J). Increased expression of the surface markers onT cells was not observed in the presence of the B7H3-targetingconstrained CD3 engager constructs in cultures with B7H3 negative celllines (FIGS. 11E and 11G for CD4+ T cells and FIGS. 11I and 11K for CD8+T cells).

D. T Cell Cytokine Production

Supernatants from T cell cytotoxicity tumor cell co-culture assays,involving co-culture of T cells with B7H3 positive, A375 or negative,CCRF-CEM cells in the presence of cx5952, cx6083, cx6084 or cx5067, wereanalyzed for IFNγ content by sandwich ELISA. A standard curve wasgenerated from which cytokine concentration values of supernatantsamples were interpolated. Samples that had absorbance values below thelower limit of detection were assigned a cytokine concentration equal tohalf that of the lowest standard concentration. As shown in FIG. 12A,the representative sdAb-Fc-dsFV-sdAb construct, cx5952, was superior tothe tested B7H3-targeted FAB containing constructs, cx6083, cx6084 andcx5067 at eliciting target-dependent cytokine release from activatedT-cells. Importantly, the MAB-dsFV constructs, cx6083 and cx6084 weresuperior to the MAB-FV construct, cx5067, demonstrating the importanceof interdomain disulfide stabilizing modification for enhancing T-cellfunction.

When compared to the other exemplary B7H3-targeting constrained CD3engagers with sdAb B7H3-targeting domains (cx6079, cx6080 and cx6081),the exemplary cx5952 sdAb B7H3-targeting constrained CD3 engagermediated substantially increased production of IFNγ in the presence ofB7H3-target cells T cells but not in cultures with B7H3 negative celllines (FIG. 12B)

E. Summary

These observations further support that the antigen-targeted constrainedCD3 format provided herein lack or exhibit reduced T-cell binding inisolation while maintaining potent B7H3-dependent T-cell cytotoxicityinducing capacities. Without wishing to be bound by theory, togetherthese results show that utilization of antigen targeted sdAbs instead ofa Fabs may reduce the immune synapse distance between the TAA expressingtumor cell and the CD3 expressing T-cells and enhance T cell activityand cytotoxicity. Notably, it was found that the inclusion of aninterchain disulfide bond created by the modification of anti-CD3 VHG44C paired with VL G100C greatly enhanced the activity of constrainedCD3 engaging constructs. Further, the more potent B7H3-dependent T cellactivity by cx5952 compared to other sdAb B7H3-targeting domainconstructs suggests that the positioning of the B7H3-targeting sdAbC-terminal to the anti-CD3 binding domain or the fact that cx5952 bindstwo distinct epitopes on B7H3 whereas the other constructs tested bindto a single epitope in a bivalent manner, contributed to this enhancedactivity.

Example 5: Assessment of CD3-Constrained Multispecific ConstructsContaining Single or Multiple B7H3-Binding Targeting Domains

Activity of constructs containing a monovalent sdAb antigen-bindingdomain (positioned at either the N or C-terminus) was compared toactivity of dual binding (bivalent) constructs that containedantigen-targeting sdAbs positioned at both the N and C-termini. Bindingwas assessed substantially as described in Example 2 and T cell activitywas assessed in the Jurkat reporter assay and T cell cytotoxicity assayssubstantially as described in Examples 3 and 4.

A. Binding

As shown in FIG. 13A the bivalent B7H3-targeting constrained CD3engaging constructs, cx5187 and cx5823, displayed higher affinitybinding to B7H3 positive A375 cells, compared to the monovalentversions, cx5873 and cx5965. None of these constructs displayed anydetectable binding to B7H3 negative CCRF-CEM cells or isolated T-cells(FIG. 13B).

B. T Cell Reporter Activity

B7H3 Antigen-dependent CD3 agonistic capacities of antigen-targetedconstrained CD3 engaging constructs that engage the antigen in amonovalent or bivalent manner were assessed using CD3-NFAT Jurkatreporter cells, in an assay substantially as described above. As shownin FIG. 13C, substantially increased fluorescence reporter activity wasobserved in the presence of the exemplary bivalent B7H3-targetedconstruct cx5187 compared to reporter activity for the exemplarymonovalent constructs cs5873 and cx5965. No reporter activity wasobserved when constructs were incubated with Jurkat reporter cellsco-cultured with B7H3-negative CCRF target cells (FIG. 13D).

C. Cytotoxic Activity

Cytotoxicity of B7H3-targeted CD3 constrained binding constructs wasassessed against a melanoma cell line, A375, and a T-cell acutelymphoblastic leukemia cell line, CCRF-CEM, which were used as B7H3positive and negative cell lines, respectively. Cytotoxicity wasassessed substantially as described in Example 4. As shown in FIG. 14Aan exemplary bivalent B7H3-targeted constrained CD3 engaging construct,cx5187, displayed enhanced target-dependent T-cell mediated cytotoxicitycompared to the monovalent versions of the constructs, cx5873 andcx5965. In these assays, no cytotoxicity was observed in the absence ofB7H3 expression of the target cells, as shown in FIG. 14B wherein theCCRF-CEM cells were used as target cells.

D. T Cell Modulation

T cell modulation was assessed by monitoring expression of CD25,substantially as described in Example 4, in suspension cells from T cellcytotoxicity assays above, involving culture of T cells with B7H3positive (A375) or B7H3 negative cell lines (CCRF-CEM) in the presenceof cx5187, cx5873 or cx5965. As shown in FIGS. 15A and 15B, an exemplarybivalent B7H3-targeted constrained CD3 engaging construct, cx5187,displayed enhanced target-dependent T-cell mediated activation comparedto the monovalent versions of the constructs, cx5873 and cx5965, asevidenced by enhanced potency of CD25 upregulation on CD4 and CD8T-cells. In these assays, no T-cell activation was observed in theabsence of B7H3 expression of the target cells, as shown in FIGS. 15Cand 15D, wherein the CCRF-CEM cells were used as target cells. Theseresults demonstrated that the B7H3-targeting constrained CD3 engagingconstructs induced potent antigen-dependent activation of both CD4 andCD8 T-cells.

D. Summary

Together, these results demonstrate that bivalent antigen-targetedconstrained CD3 engaging constructs displayed superior antigen-dependentCD3 binding and activity than the monovalent antigen-targetedconstrained CD3 engaging constructs. These results are consistent with afinding that constructs containing dual antigen-binding domainspositioned at both the N and C-termini have superior binding and T cellactivity than monovalent constructs containing only a single monovalentantigen-binding domain. Furthermore, without wishing to be bound bytheory, positioning one of the sdAbs C-terminal to the CD3 bindingdomain may form a more optimal immune synapse compared to constructswherein the sdAbs are only positioned N-terminal to the Fc as the lattermay increase the immune synapse distance.

Example 6: Assessment of a CD3-Constrained Multispecific ConstructsContaining B7I13-Targeting sdAb and Fab Domains

Constructs containing either B7H3-targeted sdAb(s) or a Fab as thetumor-associated antigen-binding domain were assessed for T-cellactivating activity. Activity of B7H3-targeted constrained CD3 engagingconstructs that were formatted with anti-B7H3 sdAbs (e.g. cx5952 andcx6079) or anti-B7H3 MAB constructs formatted with a Fab (e.g. cx5067,cx6083 or cx6084) as the antigen-binding domain(s) were assessed (seeFIGS. 3A and 3C and Table E1.1 and E1.2). All tested constructs, exceptcx6079 and cx5067, contained a disulfide-stabilized anti-CD3 Fv (dsFv)containing an interchain disulfide bond created by the modification ofanti-CD3 VH G44C paired with VL G100C. The anti-CD3 Fv of cx5067,designated MAB-Fv, and the anti-CD3 Fv of cx6079, designated sdAb-Fc-Fv,were not disulfide-stabilized. Additionally, cx5952 was engineered tocontain two distinct B7H3-targeting sdAb domains, with one locatedN-terminal to the Fc domain and one located C-terminal to theCD3-binding domain. By contrast, cx6079 was engineered to contain twoidentical B7H3-targeting sdAb domains, both located N-terminal to the Fcdomain. The Fvs of all three Fab constructs were engineered to beN-terminal to the Fc domain.

A. Cytotoxicity

Cytotoxicity of B7H3-targeted CD3 constrained binding constructs wasassessed substantially as described in Example 4. Cytotoxicity wasassessed against a melanoma cell line, A375, and a T-cell acutelymphoblastic leukemia cell line, CCRF-CEM, which were used as B7H3positive and negative cell lines, respectively. As shown in FIG. 16A theexemplary constrained CD3 engaging constructs formatted withB7H3-targeting sdAbs, cx5952 and cx6079, were superior at elicitingantigen-dependent T-cell cytotoxicity compared to the anti-B7H3 MABconstructs formatted with a Fab, cx5067, cx6083, and cx6084. Notably,cx5952 was more potent than cx6079, suggesting the positioning of theB7H3-targeting sdAb C-terminal to the anti-CD3 binding domain and/or thestabilization of the anti-CD3 FV via engineered disulfide contributed tothis enhanced activity. In these assays, no cytotoxicity was observed inthe presence of B7H3-negative CRF-CEM cells target cells, as shown inFIG. 16B.

B. T Cell Modulation

To further assess T cell modulation, exemplary multispecific CD3constrained binding constructs were assessed by monitoring the abilityof the constructs to modulate T cell activation markers, substantiallyas described in Example 4. To assess T cell activation, suspension cellsfrom T cell cytotoxicity assays above, involving culture of T cells withB7H3 positive (A375) or B7H3 negative cell lines (CCRF-CEM) in thepresence of an exemplary B7H3-targeted constrained CD3 engagingconstructs, were collected. Tested constructs included anti-B7H3constructs formatted with sdAbs (e.g. cx5952 and cx6079) and anti-B7H3constructs formatted with a Fab (e.g. cx5067, cx6083 and cx6084)

Cells were stained with a live/dead stain and fluorophore-conjugatedanti-CD4, anti-CD8, anti-CD25 and/or anti-CD71 antibodies. Cells wereanalyzed using a SONY SA3800 spectral analyzer and CD4+ or CD8+ T cellactivation was determined by measuring expression levels of CD25 or CD71or percent CD25- or CD71-positive.

Results are shown for CD25 expression (FIG. 16C-F) and CD71 expression(FIG. 16G-J) on CD4+ and CD8+ T cells following the co-culture with B7H3positive (A375) or B7H3 negative cell lines (CCRF-CEM) in the presenceof the described constructs. The results showed that cx5952 mediated adose-dependent B7H3-dependent T-cell activation via CD3 binding, asevidenced by increased expression of CD25 and CD71 in CD4+ and CD8+ Tcells. cx5952 was the most potent over other B7H3-targeted constrainedCD3 engaging constructs and inducing T-dependent T-cell activation.

C. T Cell Cytokine Production

Supernatants from T cell cytotoxicity tumor cell co-culture assays,involving co-culture of T cells with B7H3 positive, A375 or negative,CCRF-CEM cells in the presence of cx5952, cx6079, cx6083, cx6084 orcx5067, were analyzed for IFNγ content by sandwich ELISA. A standardcurve was generated from which cytokine concentration values ofsupernatant samples were interpolated. Samples that had absorbancevalues below the lower limit of detection were assigned a cytokineconcentration equal to half that of the lowest standard concentration.As shown in FIG. 16K, the representative sdAb-Fc-dsFV-sdAb construct,cx5952, was superior to the tested B7H3-targeted FAB containingconstructs, cx6083, cx6084 and cx5067 at eliciting target-dependentcytokine release from activated T-cells. This is consistent with thefinding from the antigen dependent cytotoxicity and activation assays.Importantly, the MAB-dsFV constructs, cx6083 and cx6084 were superior tothe MAB-FV construct, cx5067, demonstrating the importance ofinterdomain disulfide stabilizing modification for enhancing T-cellfunction.

D. Summary

Together, these results demonstrate that constrained anti-CD3 constructsformatted with anti-B7H3 sdAb binding domains were superior at elicitingantigen-dependent T-cell cytotoxicity compared to the anti-B7H3 MABconstructs formatted with a Fab B7H3 binding domain. Further, thatcx5952 was more potent than cx6079 suggested that the positioning of theB7H3-targeting sdAb C-terminal to the CD3 binding domain, thestabilization of the anti-CD3 FV via engineered disulfide, or both,contribute to enhanced activity. Without wishing to be bound by theory,positioning one of the sdAbs C-terminal to the CD3 binding domain mayform a more optimal immune synapse compared to constructs wherein thesdAbs are only positioned N-terminal to the Fc as the latter mayincrease the immune synapse distance.

Example 7: Assessment of CD3-Constrained Multispecific ConstructsContaining Single or Multiple Antigen-Binding DLL3-Targeting Domains

This example describes the assessment and characterization of exemplarygenerated DLL3-targeted constrained CD3 engaging constructs in humanprimary T cell in vitro assays.

Binding and activity of DLL3-targeted constrained CD3 engagingconstructs that were formatted with an anti-DLL3 sdAb (e.g. cx5352,cx5800, cx5801, and cx5499) as the antigen-binding domain(s) wereassessed (see FIGS. 4A-4B and Table E1.1). All tested constructscontained a disulfide-stabilized anti-CD3 Fv (dsFv) containing aninterchain disulfide bond created by the modification of anti-CD3 VHG44C paired with VL G100C. Further, the DLL-3-targeted constructs wereengineered to contain a co-stimulatory receptor sdAb C-terminal to theCD3 dsFv, except for cx5499, which did not contain this co-stimulatoryreceptor sdAb domain.

A. Binding

Binding was assessed substantially as described in Example 2. As shownin FIG. 17A the bivalent DLL3-targeting constrained CD3 engagingconstructs, cx5352 displayed higher affinity binding to DLL3 positive,SHP-77 cells compared to the monovalent versions, cx5800 and cx5801.None of the constructs tested displayed binding to DLL3-negative primaryT cells, as depicted in FIG. 17B. These binding assays were conducted byflow cytometry, wherein bound constructs were detected using afluorophore-conjugated anti-human IgG Fc secondary antibody.

A. T Cell Reporter Activity

T cell activity was assessed in a reporter assay substantially asdescribed in Example 2, except that Jurkat cells expressing NFAT-drivenLuciferase were used and luciferase activity was monitored. NFAT-drivenLuciferase CD3 Jurkat reporter cells were co-cultured with SHP-77(DLL3-positive) target cells in the presence of monovalent and bivalentconstructs containing antigen-binding domains against the DLL3 antigen(see FIG. 4A). Specifically, as shown in FIG. 17C, the exemplarybivalent construct cx5352 induced substantially greater luciferaseactivity in this assay compared to the exemplary monovalent constructscx5800 and cx5801. These results are consistent with results observedwith B7H3-targeted constructs, thereby indicating that the activity ofthe constructs is not specific to a particular target antigen.

C. Cytotoxicity

Cytotoxicity of cx5499, a DLL3-targeted CD3 constrained bindingconstruct formatted with two distinct sdAb binding domains located atits amino and carboxy termini, was assessed against a DLL3 expressingcell line, SHP-77, using an assay substantially as described in Example4. As shown in FIG. 18A, cx5499 induced potent T-cell mediatedcytotoxicity directed toward the SHP-77 cell line.

D. T Cell Modulation

To further assess T cell modulation, exemplary multispecific CD3constrained binding constructs were assessed by monitoring the abilityof the constructs to modulate T cell activation markers. To assess Tcell activation, suspension cells from T cell cytotoxicity assays above,involving culture of T cells with DLL3 positive SHP-77 cells in thepresence of cx5499, in the presence of an exemplary DLL3-targetedconstrained CD3 engaging constructs, were collected. Cells were stainedwith a live/dead stain and fluorophore-conjugated anti-CD4, anti-CD8,anti-CD25 and/or anti-CD69 antibodies. Cells were analyzed using a SONYSA3800 spectral analyzer and CD4+ or CD8+ T cell activation wasdetermined by measuring expression levels of CD25 or CD69 or percentCD25- or CD69-positive.

FIG. 18B and FIG. 18D depict results for CD25 expression on CD4 T cellsor CD8 T cells, respectively, upon culture of T cells with DLL3positive, SHP-77 cells, in the presence of an exemplary DLL3-targetedconstrained CD3 engaging construct, cx5499. FIG. 18C and FIG. 18E depictresults for CD69 expression on CD4 cells or CD8 T cells, respectively,upon culture of T cells with DLL3 positive, SHP-77 cells, in thepresence of an exemplary DLL3-targeted constrained CD3 engagingconstruct, cx5499. The results showed that cx5499 mediated adose-dependent DLL3-dependent T-cell activation via CD3 binding, asevidenced by increased expression of CD25 and CD69 on CD4+ and CD8+ Tcells.

E. Summary

Together, these results demonstrate that constrained anti-CD3 constructsformatted with anti-DLL3 sdAb binding domains are capable of binding toa DLL3-expressing cell line, SHP-77, and eliciting antigen-dependentT-cell cytotoxicity and activation. This result is consistent with afinding that the constrained CD3 engaging constructs of the disclosurehave broad applicability to specifically target numerous tumor antigensand elicit T-cell cytotoxicity and activation against target-expressingcells.

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the disclosure, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

SEQUENCE TABLE SEQ ID DESCRIPTION NO SEQUENCE 1PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV IgG1 FcDGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEYKCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLVKGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQGNVFSCSVMH EALHNHYTQK SLSLSPGK 2PAPGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV xELL FcEVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCKVSNKALPAPI EKTISKAKGQ PREPQVYTLP PSRDELTKNQ VSLTCLVKGFYPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTVDKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK 3PAPPVAGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFNWYVD IgG2 FcGVEVHNAKTK PREEQFNSTF RVVSVLTVVH QDWLNGKEYKCKVSNKGLPA PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVKGFYPSDISVE WESNGQPENN YKTTPPMLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE ALHNHYTQKS LSLSPGK 4PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVQFKWYV IgG3 FcDGVEVHNAKT KPREEQYNST FRVVSVLTVL HQDWLNGKEYKCKVSNKALP APIEKTISKT KGQPREPQVY TLPPSREEMT KNQVSLTCLVKGFYPSDIAV EWESSGQPEN NYNTTPPMLD SDGSFFLYSK LTVDKSRWQQGNIFSCSVMH EALHNRFTQK SLSLSPGK 5PAPEFLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSQE DPEVQFNWYV IgG4 FcDGVEVHNAKT KPREEQFNST YRVVSVLTVL HQDWLNGKEYKCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLVKGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQEGNVFSCSVMH EALHNHYTQK SLSLSLGK 6PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSQE DPEVQFNWYV IgG4 FcDGVEVHNAKT KPREEQFNST YRVVSVLTVL HQDWLNGKEYKCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLVKGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQEGNVFSCSVMH EALHNHYTQK SLSLSLGK 7 EPKSSDKTHTCPPC Hinge 8 DKTHTCPPC Hinge9 ESKYGPPCPPC Hinge 10 GGSGGS (GGS)₂ 11 GGSGGSGGS (GGS)₃ 12 GGSGGSGGSGGS(GGS)₄ 13 GGSGGSGGSGGSGGS (GGS)₅ 14EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVA anti-CD3 VHRIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSA 15QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIG anti-CD3 VLGTNKRAPGVPARFSGSLIGDKAALTITGAQIEDEAIYFCALWYSNLWVFGG GTKLTVL 16 TYAMNanti-CD3 VH CDR1 17 RIRSKYNNYATYYADSVKD anti-CD3 VH CDR2 18HGNFGNSYVSWFAY anti-CD3 VH CDR3 19 RSSTGAVTTSNYAN anti-CD3 VL CDR1 20GTNKRAP anti-CD3 VL CDR2 21 ALWYSNLWV anti-CD3 VL CDR3 22 LEADGranzyme B substrate 23 RQAR Granzyme B substrate 24 PAGL MMP substrate25 TGLEADGSPAGLGRQARVG Linker 26 TGLEADGSRQARVGPAGLG Linker 27TGSPAGLEADGSRQARVGS Linker 28 TGPAGLGLEADGSRQARVG Linker 29TGRQARVGLEADGSPAGLG Linker 30 TGSRQARVGPAGLEADGS Linker 31TGPAGLGSRQARVGLEADGS Linker 32EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGR anti-CD3 VH1IRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 33EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVA anti-CD3 VH2RIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 34EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVAR anti-CD3 VH3IRSKYNNYATYYADSVKDRFTISRDDSKSILYLQMNNLKIEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 35EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVAR anti-CD3 VH4IRSKYNNYATYYADSVKDRFTISRDDSKSILYLQMNSLKIEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 36EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVAR anti-CD3 VHSIRSKYNNYATYYADSVKDRFTISRDDSKSILYLQMNSLKIEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 37EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRI anti-CD3 VH6RSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 38EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVGR anti-CD3 VH7IRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGDSYVSWFAYWGQGTLVTVSS 39EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVA anti-CD3 VH8RIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVS 40EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVA anti-CD3 VH9RIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTTVTVSS 41EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVA anti-CD3 VH10RIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSYFAYWGQGTTVTVSS 42EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVA anti-CD3 VH11RIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 43EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVA anti-CD3 VH12RIRSKYNNYATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVKP 44EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVAR anti-CD3 VH13IRSKYNNYATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVKP 45EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVA anti-CD3 VH14RIRSKYNNYATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGCGTLVTVKP 46EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVAR anti-CD3 VH15IRSKYNNYATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 47EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVSR anti-CD3 VH16IRSKYNNYATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 48EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSR anti-CD3 VH17IRSKYNNYATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 49EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKCLEWVAR anti-CD3 VH18IRSKYNNYATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 50EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVSR anti-CD3 VH19IRSKYNNYATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 51EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKCLEWVSRI anti-CD3 VH20RSKYNNYATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 52EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKCLEWVGR anti-CD3 VH21IRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 53EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVAR anti-CD3 VH22IRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 54EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVAR anti-CD3 VH23IRSKYNNYATYYADSVKDRFTISRDDSKSILYLQMNNLKIEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 55EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVAR anti-CD3 VH24IRSKYNNYATYYADSVKDRFTISRDDSKSILYLQMNSLKIEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 56EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVAR anti-CD3 VH25IRSKYNNYATYYADSVKDRFTISRDDSKSILYLQMNSLKIEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 57EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKCLEWVSRI anti-CD3 VH26RSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 58EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMSWVRQAPGKCLEWVGRI anti-CD3 VH27RSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGDSYVSWFAYWGQGTLVTVSS 59EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKCLEWVA anti-CD3 VH28RIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVS 60EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVAR anti-CD3 VH29IRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTTVTVSS 61EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVAR anti-CD3 VH30IRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSYFAYWGQGTTVTVSS 62EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKCLEWVA anti-CD3 VH31RIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 63QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIG anti-CD3 VL1GTNKRAPGVPARFSGSLIGDKAALTITGAQIEDEAIYFCALWYSNLWVFGG GTKLTVL 64QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGKSPRGLIG anti-CD3 VL2GTNKRAPGVPARFSGSLLGGKAALTISGAQPEDEADYYCALWYSNHWVFG CGTKLEIK 65QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIG anti-CD3 VL3GTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFG GGTKLTVL 66QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIG anti-CD3 VL4GTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALWYSNLWVFGG GTKLTVL 67QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIG anti-CD3 VL5GTNKRAPGVPARFSGSILGNKAALTITGAQADDESIYFCALWYSNLWVFGG GTKLTVL 68QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIG anti-CD3 VL6GTNKRAPGVPARFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFG GGTKLTVL 69QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIG anti-CD3 VL7GTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFG GGTKLTVL 70QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIG anti-CD3 VL8GTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFG GGTKLTVL 71QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAFRGLIG anti-CD3 VL9GTNKRAPGVPARFSGSLLGGKAALTISGAQPEDEADYYCALWYSNHWVFG GGTKLEIK 72QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAFRGLIG anti-CD3 VL10GTNKRAPGVPARFSGSLLGGKAALTISGAQPEDEADYYCALWYSNHWVFG CGTKLEIK 73QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQCFRGLIG anti-CD3 VL11GTNKRAPGVPARFSGSLLGGKAALTISGAQPEDEADYYCALWYSNHWVFG EGTKLEIK 74QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIG anti-CD3 VL12GTNKRAPGVPARFSGSLIGDKAALTITGAQIEDEAIYFCALWYSNLWVFGC GTKLTVL 75QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGKSPRGLIG anti-CD3 VL13GTNKRAPGVPARFSGSLLGGKAALTISGAQPEDEADYYCALWYSNHWVFG GGTKLEIK 76QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIG anti-CD3 VL14GTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFG CGTKLTVL 77QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIG anti-CD3 VL15GTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALWYSNLWVFGG GTKLTVL 78QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIG anti-CD3 VL16GTNKRAPGVPARFSGSILGNKAALTITGAQADDESIYFCALWYSNLWVFGC GTKLTVL 79QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIG anti-CD3 VL17GTNKRAPGVPARFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFG CGTKLTVL 80QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIG anti-CD3 VL18GTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFG CGTKLTVL 81QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIG anti-CD3 VL19GTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFG CGTKLTVL 82DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Knob FcKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPT 83DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMRSRTPEVTCVVVDVSHEDPE Hole FcVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPT 84DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF Knob FcNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPT85 DKTHTCPPCPAPGGPSVFLFPPKPKDTLMRSRTPEVTCVVVDVSHEDPEVKF Hole FcNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPT86 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Knob FcKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 87DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMRSRTPEVTCVVVDVSHEDPE Hole FcVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 88DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF Knob FcNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG89 DKTHTCPPCPAPGGPSVFLFPPKPKDTLMRSRTPEVTCVVVDVSHEDPEVKF Hole FcNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG90 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Hole FcKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCS VMHEALHNRYTQKSLSLSPT91 DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF Hole FcNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSV MHEALHNRYTQKSLSLSPT92 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Hole FcKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCS VMHEALHNRYTQKSLSLSPG93 DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF Hole FcNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSV MHEALHNRYTQKSLSLSPG94 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEV Knob FcKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVVHEALHNHYTQKSLSLSPT 95DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFN Knob FcWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVV HEALHNHYTQKSLSLSPT96 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEV Knob FcKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVVHEALHNHYTQKSLSLSPG 97DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFN Knob FcWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVV HEALHNHYTQKSLSLSPG98 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEV Hole FcKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCS VVHEALHNRYTQKSLSLSPT99 DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFN Hole FcWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVVH EALHNRYTQKSLSLSPT100 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEV Hole FcKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCS VVHEALHNRYTQKSLSLSPG101 DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFN Hole FcWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVVH EALHNRYTQKSLSLSPG102 PGGGG Peptide Linker 103 GGGG Peptide Linker 104 GPAGLGLEPDGSRQARVGLinker 105 GGSGGGGIEPDIGGSGGS Linker 106 GGSGGGGLEADTGGSGGS Linker 107GSIEPDIGS Linker 108 GSLEADTGS Linker 109 GGSGGGGIEPDGGGSGGS Linker 110GGSGGGGIEPDVGGSGGS Linker 111 GGSGGGGIEPDSGGSGGS Linker 112GGSGGGGIEPDTGGSGGS Linker 113 GGGSLEPDGSGS Linker 114 GPAGLGLEADGSRQARVGLinker 115 GGEGGGGSGGSGGGS Linker 116 GSSAGSEAGGSGQAGVGS Linker 117GGSGGGGLEAEGSGGGGS Linker 118 GGSGGGGIEPDPGGSGGS Linker 119GGGGGSGGGGGSGGGGGS Linker 120QLQLQESGGGLVQPGGSLRLSCAASGFTLDNYAIGWFRQAPGKEREGVSCIS FR alpha sdAbSSDGSTYYADSVKGRFTISRNNAKGTVYLLMNSLKPEDTAVYYCATELVPACTYSNGRGPLDGMDYWGKGTQVTVKP 121EVQLLESGGGEVQPGGSLRLSCAASGSIFSIDATAWYRQAPGKQRELVAIITS FR alpha sdAbSGSTNYPESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCNAITRYGGS TYDFWGQGTLVTVKP 122EVQPGGSLRLSCAASETFGVVFTLGWYRQAPGKGREFVARVTGTDTVDYA FR alpha sdAbESVKGRFTISSDFARNTVYLQMNSLRAEDTAVYYCNTGAYWGQGTLVTVK P 123EVQLVESGGGLVQPGGSLRLSCAASGFILDYYAIGWFRQAPGKEREGVLCID cMET sdAbASDDITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCATPIGLSSSCLLEYDYDYWGQGTLVTVKP 124EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAY B7H3 scFvISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSSGGGGSGGGGSGGGGSDIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIK 125QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMG CD20 scFvRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSGSGGGGSGGGGTGGGGSDIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIK 126QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYVY DLL3 scFvYSGTTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASIAVTGFYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERVTLSCRASQRVNNNYLAWYQQRPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKLEIK 127EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAY B7H3 FdISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSC128 DIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSAS B7H3 LCYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 129EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVA 5T4 FdRIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRQWDYDVRAMNYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 130DIVMTQSHIFMSTSVGDRVSITCKASQDVDTAVAWYQQKPGQSPKLLIYWA 5T4 LCSTRLTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC 131QVQLQESGPGLVKPSQTLSLTCTVSGGSISSFNYYWSWIRHHPGKGLEWIGY gpNMB FdIYYSGSTYSNPSLKSRVTISVDTSKNQFSLTLSSVTAADTAVYYCARGYNWNYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSC 132EIVMTQSPATLSVSPGERATLSCRASQSVDNNLVWYQQKPGQAPRLLIYGAS gpNMB LCTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC 133QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMA DLL3 FdWINTYTGEPTYADDFKGRFAFSLETSASTASLQIINLKNEDTATYFCARIGDSSPSDYWGQGTTLTVSSSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSC134 SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVVWYQQKPGQSPKLLIYYAS DLL3 LCNRYTGVPDRFAGSGYGTDFSFTISTVQAEDLAVYFCQQDYTSPWTFGGGTKLEIRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC 135 GGGGGSPeptide Linker 136 IEPDI Linker 137 LEADT Linker 138 IEPDG Linker 139IEPDV Linker 140 IEPDS Linker 141 IEPDT Linker 142 LEPD Linker 143 LEAELinker 144 IEPDP Linker 145QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIG SecondGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFG PolypeptideGGTKLTVLGGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGM Chain of B7-H3HWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNS x CD3 BispecificLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSGGCGGGKVAALKE DART-AKVAALKEKVAALKEKVAALKE Diabody 146DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE ThirdVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC PolypeptideKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFY Chain of B7-H3PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS x CD3 BispecificCSVMHEALHNRYTQKSLSLSPGK DART-A Diabody 147 GGSGGGGSGGGGSGGGGS Linker 148TGGSGGGGIEPDIGGSGGS Linker 149 GGGGS Linker 150X₁ X₂ X₃ X₄ X₅ (P4 P3 P2 P1 ↓ P1′) Linker consensus X1 =I, L, Y, M, F, V, or A; (P4 = I, L, Y, M, F, V, or A) X2 =A, G, S, V, E, D, Q, N, or Y; (P3 = A, G, S, V, E, D, Q, N, or Y) X3 =H, P, A, V, G, S, or T; (P2 = H, P, A, V, G, S, or T) X4 = D or E; (P1 =D or E) X5 = I, L, Y, M, F, V, T, S, G or A (P1′ = I, L, Y, M, F, V,T, S, G or A) 151 X1 E X3 D X5 (P4 P3 P2 P1 ↓ P1′) Linker consensus X1 =I or L; (P4 = I or L) (P3 = E) X3 = P or A; (P2 = P or A) X5 =I, V, T, S, or G (P1′ = I, V, T, S, or G) 152 LEPDG Linker 153 LEADGLinker 154 X₁QARX₅ (P1QAR↓(A/V)) Linker consensus X1 =any amino acid; (P1 is any amino acid) X5 = A or V 155RQARX₅ (RQAR(A/V)) Linker consensus X5 = A or V 156 RQARV Linker 157X1X2 X3 X4 (P3 P2 P1 ↓ P1′) Linker consensus X1 = P, V or A; (P3 =P, V or A) X2 = Q or D; (P2 = Q or D) X3 = A or N; (P1 = A or N) X4 =L, I or M (P1′ = L, I or M) 158 PX2X3X4 (P3P2 P1 ↓ P1′) Linker consensus(P3 = P) X2 = Q or D; (P2 = Q or D) X3 = A or N; (P1 = A or N) X4 =L or I (P1′ = L or I) 159 GSGATNFSLLKQAGDVEENPGP P2A 160ATNFSLLKQAGDVEENPGP P2A 161 QCTNYALLKLAGDVESNPGP E2A 162VKQTLNFDLLKLAGDVESNPGP F2A 163 EGRGSLLTCGDVEENPGP T2A 164LEGGGEGRGSLLTCGDVEENPGPR T2A 165GGATCTGGAGCAACAAACTTCTCACTACTCAAACAAGCAGGTGACGTGG AGGAGAATCCCGGACCCP2A DNA 166 GSPAGLEADGSRQARVGS Linker 167EVQLVESGGGL VQPKGSLKLS CAASGFTFNT YAMNWVRQAP anti-5T4 VHGKGLEWVARI RSKSNNYATY YADSVKDRFT ISRDDSQSML YLQMNNLKTE DTAMYXCVRQ WDYDVRAMNY WGQGTSVTVS S 168DIVMTQSHIF MSTSVGDRVS ITCKASQDVD anti-5T4 VLTAVAWYQQKP GQSPKLLIYW ASTRLTGVPD RFTGSGSGTD FTLTISNVQSEDLADYFCQQ YSSYPYTFGG GTKLEIK 169DIQLTQSPSF LSASVGDRVT ITCKASQNVD TNVAWYQQKP GKAPKALIYS First PolypeptideASYRYSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YNNYPFTFGQ Chain of B7-H3GTKLEIKGGG SGGGGEVQLV ESGGGLVQPG GSLRLSCAAS x CD3 Bispecific GFTFSTYAMNDART-A WVRQAPGKGL EWVGRIRSKY NNYATYYADS VKDRFTISRD DiabodyDSKNSLYLQM NSLKTEDTAV YYCVRHGNFG NSYVSWFAYWGQGTLVTVSS GGCGGGEVAA LEKEVAALEK EVAALEKEVAALEKGGGDKT HTCPPCPAPE AAGGPSVFLF PPKPKDTLMI SRTPEVTCVVVDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVVSVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQPREPQVYTLPP SREEMTKNQV SLWCLVKGFY PSDIAVEWES NGQPENNYKTTPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK 170GGGGSGGGGSGGGGS Linker 171 GGS(GGS)n Linker wherein n is 0 to 10 172(GGGGGS)n Linker wherein n is 1 to 4 173 (GGGGS)n Linkerwherein n is 1 to 10 174 Gly_(x)Xaa-Gly_(y)-Xaa-Gly_(z) LinkerXaa is independently selected from A, V, L, I, M, F, W, P,G, S, T, C, Y, N, Q, K, R, H, D, or Ex, y, and z are each integers in the range from 1-5 175Gly-Gly-Gly-Xaa-Gly-Gly-Gly-Xaa-Gly-Gly-Gly LinkerXaa is independently selected from A, V, L, I, M, F, W, P,G, S, T, C, Y, N, Q, K, R, H, D, or E 176 ATTTGSSPGPT Linker 177GGGGG-C-GGGGG Linker 178 (EAAAK)n Linker n = 2-20 179 AS-(AP)n-GT Linkern = 2-20 180 AS-(EAAAK)n-GT Linker n = 2-20 181 (GGGGA)n Linker n = 2-20182 (PGGGS)n Linker n = 2-20 183 (AGGGS)n Linker n = 2-20 184GGS-(EGKSSGSGSESKST)n-GGS Linker n = 2-20 185 (SSSSG)n Linker n = 1-9186 SSSASASSA Linker 187 GSPGSPG Linker 188QVQLQESGPG LVKPSETLSL TCTVSGGSIS SYYWSWIRQP PGKGLEWIGY DLL3 scFvVYYSGTTNYN PSLKSRVTIS VDTSKNQFSL KLSSVTAADT AVYYCASIAVTGFYFDYWGQ GTLVTVSSGG GGSGGGGSGG GGSEIVLTQS PGTLSLSPGERVTLSCRASQ RVNNNYLAWY QQRPGQAPRL LIYGASSRAT GIPDRFSGSGSGTDFTLTIS RLEPEDFAVY YCQQYDRSPL TFGGGTKLEI K 189QVQLVQSGAE VKKPGSSVKV SCKASGYAFS YSWINWVRQA CD20 VH PGQGLEWMGRIFPGDGDTDY NGKFKGRVTI TADKSTSTAY MELSSLRSED TAVYYCARNVFDGYWLVYWG QGTLVTVSS 190DIVMTQTPLS LPVTPGEPAS ISCRSSKSLL HSNGITYLYW YLQKPGQSPQ CD20 VLLLIYQMSNLV SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCAQNLELP YTFGGGTKVE IKRTV191 QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAFRGLIG anti-CD3 VL35GTNKRAPGVPARFSGSLLGGKAALTISGAQPEDEADYYCALWYSNHWVFG (CON) CGTKLTVL 192GGGGG linker 193 GGGGSGGGGSGGGGS linker 194DKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEV Fc-Het-1KFNWYVDGVEVHNAKTKPREEEYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCDVSGFYPSDIAVEWESDGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWEQGDVFSCSVMHEALHNHYTQKSLSLSPGK 195DKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEV Fc-Het-2KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREQMTKNQVKLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 196EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGR CD3-VH32IRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGDSYVSWFAYWGQGTLVTVSS 197EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKCLEWVGR CD3-VH33IRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGDSYVSWFAYWGQGTLVTVSS 198EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKCLEWVAR CD3-VH34IRSKYNNYATYYADTVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVRHGNFGDSYVSWFAYWGQGTLVTV 199QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGKSPRGLIG CD3-VL20GTNKRAPGVPARFSGSLLGGKAALTISGAQPEDEADYYCALWYSNHWVFG GGTKLTVL 200QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGKSPRGLIG CD3-VL21GTNKRAPGVPARFSGSLLGGKAALTISGAQPEDEADYYCALWYSNHWVFG CGTKLTVL 201DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Knob FcKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSP202 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMRSRTPEVTCVVVDVSHEDPE Hole FcVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSP203 DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF Knob FcNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSP204 DKTHTCPPCPAPGGPSVFLFPPKPKDTLMRSRTPEVTCVVVDVSHEDPEVKF Hole FcNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSP205 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Hole FcKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCS VMHEALHNRYTQKSLSLSP206 DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF Hole FcNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSV MHEALHNRYTQKSLSLSP207 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEV Knob FcKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVVHEALHNHYTQKSLSLSP208 DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFN Knob FcWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVV HEALHNHYTQKSLSLSP209 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEV Hole FcKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCS VVHEALHNRYTQKSLSLSP210 DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFN Hole FcWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVVH EALHNRYTQKSLSLSP 211GFTFNTYAMN anti-CD3 VH CDR1 212 RIRSKYNNYATY anti-CD3 VH CDR2 213QVQLVQSGAE VKKPGSSVKV SCKASGYAFS YSWINWVRQA CD20 scFvPGQGLEWMGR IFPGDGDTDY NGKFKGRVTI TADKSTSTAY MELSSLRSEDTAVYYCARNV FDGYWLVYWG QGTLVTVSSG GGGSGGGGSGGGGSDIVMTQ TPLSLPVTPG EPASISCRSS KSLLHSNGIT YLYWYLQKPGQSPQLLIYQM SNLVSGVPDR FSGSGSGTDF TLKISRVEAE DVGVYYCAQN LELPYTFGGG TKVEIK214 EVQLVESGGGEVQPGGSLRLSCAASGFSFSSNVMMWVRQAPGKGLEWVSTIYSSG B7H3 sdAbTGTFYAESVKGRFTISRDNAKNTLYLQMSSLRPEDTAVYYCATSGPVRGWGPRSQGT B7h3 hz1A5v51LVTVKP 215 EVQLVESGGGEVQPGGSLRLSCAASGSTFSSYHMSWFRQAPGKQREPVATS sdAb B7H3HHGGTTNYAGSVKGRFTISRDNAKNTVYLQMNTLRAEDTAVYYCKADHG hz58E05v27YQGRGYWGQGTLVTVKP 216EVQLVESGGGEVQPGGSLRLSCAASGFTFSSYHMSWFRQAPGKQRELVATSHHGGT sdAb B7H3TNYAGSVKGRFTISRDNAKNTVYLQMNTLRAEDTAVYYCKADHGYQGRGYWGQGT hz58E05v55LVTVKP 217 EVQLVESGGGEVQPGGSLRLSCAASGFTFSSYHMSWFRQAPGKQREPVATS sdAb B7H3HHGGTTNYAGSVKGRFTISRDNAKNTVYLQMNTLRAEDTAVYYCKADHG hz58E05v48YQGRGYWGQGTLVTVKP 218EVQLVESGGGEVQPGGSLRLSCAPSERTFSTYTMGWFRQAPGKEREFVAVV sdAb B7H3NWGGGSKYYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAAGG hz57B04v24AYSGPYYDTRQYTYWGQGTLVTVKPGG 219EVQLVESGGGEVQPGGSLRLSCAASGSIFSINAMGWYRQAPGKQRELVAGF sdAb DLL3TGDTNTIYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAADVQLF hz10D9v7SRDYEFYWGQGTLVTVKP 220EVQLVESGGGEVQPGGSLRLSCGPSEIITSDKSMGWVRQAPGKQRNLVAGIS sdAb DLL3NVGSTNYAQSVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCYARDFEN hz8E7v16EYWGQGTLVTVKP 221 GFSFSINAMG 41BB CDR1 222 AIESGRNTV 41BB CDR2 223LKGNRVVSPSVAY 41BB CDR3 224 HGNFGDSYVSWFAY CD3-VH7, VH33 CDR3 225ALWYSNHWV CD3-VL2, VL21 CDR3 226 VLWYSNRWV CD3-VL8 CDR3 227 GFTFSTYAMNCD3 VH33 CDR1 228 RIRSKYNNYATY CD3 VH33 CDR1 229 GSSTGAVTTSNYAN CD3 VL21CDR1 230 GTNKRAP CD3 VL21 CDR2

What is claimed:
 1. A multispecific polypeptide construct, themultispecific polypeptide construct comprising a first componentcomprising an immunoglobulin Fc region and a second component comprisinga CD3-binding region, wherein: the CD3 binding region is an anti-CD3antibody or antigen binding fragment that is an Fv antibody fragmentcomprising a variable heavy chain region (VH) and a variable light chainregion (VL); the Fc is a heterodimeric Fc comprising a first Fcpolypeptide and a second Fc polypeptide and the VH and VL of theanti-CD3 antibody or antigen binding fragment are linked to oppositepolypeptides of the heterodimeric Fc; the first and second componentsare coupled by a non-cleavable linker, wherein the Fc region ispositioned N-terminal to the CD3-binding region; and the first componentcomprises a first antigen binding domain and the second componentcomprises a second antigen binding domain, wherein each of the antigenbinding domains bind a tumor associated antigen (TAA).
 2. (canceled) 3.The multispecific polypeptide construct of claim 1, wherein the firstantigen binding domain is positioned amino-terminally relative to the Fcregion of the multispecific polypeptide construct and the second antigenbinding domain is positioned carboxy-terminally relative to the CD3binding region of the multispecific polypeptide construct.
 4. Themultispecific polypeptide construct of claim 1, wherein themultispecific polypeptide construct comprises in order, from N-terminusto C-terminus: the first antigen binding domain that binds to atumor-associated antigen (TAA); the immunoglobulin Fc region; thenon-cleavable linker; the CD3 binding region, wherein the CD3 bindingregion binds CD3 (CD3ε); and the second antigen binding domain thatbinds a tumor-associated antigen (TAA).
 5. A multispecific polypeptideconstruct, the multispecific polypeptide construct comprising a firstcomponent comprising an immunoglobulin Fc region and a second componentcomprising a CD3-binding region, wherein: the CD3 binding region is ananti-CD3 antibody or antigen binding fragment that is adisulfide-stabilized Fv antibody fragment (dsFv) comprising a variableheavy chain (VH) and a variable light chain (VL); the Fc is aheterodimeric Fc comprising a first Fc polypeptide and a second Fcpolypeptide and the VH and VL of the anti-CD3 antibody or antigenbinding fragment are linked to opposite polypeptides of theheterodimeric Fc; the first and second components are coupled by anon-cleavable linker, wherein the Fc region is positioned N-terminal tothe CD3-binding region; and one or both of the first and secondcomponents comprises an antigen binding domain that binds a tumorassociated antigen (TAA).
 6. A multispecific polypeptide construct, themultispecific polypeptide construct comprising a first componentcomprising an immunoglobulin Fc region and a second component comprisinga CD3-binding region, wherein: the CD3 binding region is an anti-CD3antibody or antigen binding fragment that is a an Fv antibody fragmentcomprising a variable heavy chain (VH) and a variable light chain (VL);the Fc is a heterodimeric Fc comprising a first Fc polypeptide and asecond Fc polypeptide and the VH and VL of the anti-CD3 antibody orantigen binding fragment are linked to opposite polypeptides of theheterodimeric Fc; the first and second components are coupled by anon-cleavable linker, wherein the Fc region is positioned N-terminal tothe CD3-binding region; and one or both of the first and secondcomponents comprises an antigen binding domain that binds a tumorassociated antigen (TAA), wherein the antigen-binding domain is a singlechain antibody fragment.
 7. The multispecific polypeptide construct ofclaim 6, wherein the single chain antibody fragment is a single domainantibody or is a single chain variable fragment (scFv).
 8. (canceled) 9.The multispecific polypeptide construct of claim 5, wherein themultispecific polypeptide construct is selected from: (A) amultispecific polypeptide construct that comprises in order, fromN-terminus to C-terminus: the first antigen binding domain that binds toa tumor-associated antigen (TAA); the immunoglobulin Fc region; thenon-cleavable linker; the CD3 binding region, wherein the CD3 bindingregion binds CD3 (CD3ε); and the second antigen binding domain thatbinds a tumor-associated antigen (TAA); or (B) a multispecificpolypeptide construct that comprises in order, from N-terminus toC-terminus: the immunoglobulin Fc region; the non-cleavable linker; theCD3 binding region, wherein the CD3 binding region binds CD3 (CD3ε); andan antigen binding domain that binds a tumor-associated antigen (TAA);or (C) a multispecific polypeptide construct that comprises in order,from N-terminus to C-terminus: the antigen binding domain that binds toa tumor-associated antigen (TAA); the immunoglobulin Fc region; thenon-cleavable linker; and the CD3 binding region, wherein the CD3binding region binds CD3 (CD3ε).
 10. The multispecific polypeptideconstruct of claim 6, wherein the multispecific polypeptide construct isselected from: (A) a multispecific polypeptide construct that comprisesin order, from N-terminus to C-terminus: the first antigen bindingdomain that binds to a tumor-associated antigen (TAA); theimmunoglobulin Fc region; the non-cleavable linker; the CD3 bindingregion, wherein the CD3 binding region binds CD3 (CD3ε); and the secondantigen binding domain that binds a tumor-associated antigen (TAA); or(B) a multispecific polypeptide construct that comprises in order, fromN-terminus to C-terminus: the immunoglobulin Fc region; thenon-cleavable linker; the CD3 binding region, wherein the CD3 bindingregion binds CD3 (CD3ε); and an antigen binding domain that binds atumor-associated antigen (TAA); or (C) a multispecific polypeptideconstruct that comprises in order, from N-terminus to C-terminus: theantigen binding domain that binds to a tumor-associated antigen (TAA);the immunoglobulin Fc region; the non-cleavable linker; and the CD3binding region, wherein the CD3 binding region binds CD3 (CD3ε). 11-14.(canceled)
 15. The multispecific polypeptide construct of claim 6,wherein the heterodimeric Fc region comprises at least one modificationselected from among the following: a steric modification(s), aknob-into-hole modification(s), a charge mutation(s) to increaseelectrostatic complementarity of the polypeptides, a modification(s) toalter the isoelectric point (pI variant), or combinations thereof.16-35. (canceled)
 36. The multispecific polypeptide construct of claim6, wherein the linker is a polypeptide linker.
 37. (canceled)
 38. Themultispecific polypeptide construct of claim 36, wherein the linker is apolypeptide of from or from about 2 to 24 amino acids. 39-43. (canceled)44. The multispecific polypeptide construct of claim 6, wherein thenon-cleavable linker comprises: (GGS)n, wherein n is 1 to 10; (GGGGS)n(SEQ ID NO: 173), wherein n is 1 to 10; or (GGGGGS)n (SEQ ID NO:172),wherein n is 1 to
 4. 45. (canceled)
 46. (canceled)
 47. The multispecificpolypeptide construct of claim 6, wherein the non-cleavable linker is orcomprises an amino acid sequence selected from among GGS;(SEQ ID NO: 149) GGGGS; (SEQ ID NO: 135) GGGGGS; (SEQ ID NO: 10) (GGS)₂; (SEQ ID NO: 11) GGSGGSGGS; (SEQ ID NO: 12) GGSGGSGGSGGS;(SEQ ID NO: 13) GGSGGSGGSGGSGGS; (SEQ ID NO: 119) GGGGGSGGGGGSGGGGGS;(SEQ ID NO: 147) GGSGGGGSGGGGSGGGGS; and (SEQ ID NO: 170)GGGGSGGGGSGGGGS.

48-56. (canceled)
 57. The multispecific polypeptide construct of claim6, wherein the multispecific polypeptide construct comprises at least(i) a first polypeptide comprising the first Fc polypeptide of theheterodimeric Fc region, the linker and the VH domain of the anti-CD3antibody or antigen binding fragment thereof; and (ii) a secondpolypeptide comprising the second Fc polypeptide of the heterodimeric Fcregion, the linker and the VL domain of the anti-CD3 antibody or antigenbinding fragment thereof, wherein one or both of the first and secondpolypeptide comprise at least one antigen-binding domain that binds to atumor associated antigen (TAA).
 58. The multispecific polypeptideconstruct of claim 6, wherein the VH of the anti-CD3 antibody orantigen-binding fragment is on the same polypeptide as the at least oneantigen-binding domain that binds to a tumor associated antigen (TAA).59. The multispecific polypeptide construct of claim 58, wherein thepolypeptide comprising the VL of the anti-CD3 antibody orantigen-binding fragment does not contain the at least oneantigen-binding domain that binds to a tumor associated antigen (TAA).60. (canceled)
 61. (canceled)
 62. The multispecific polypeptideconstruct of claim 6, wherein only one of the first and secondpolypeptide comprises the at least one antigen-binding domain that bindsa TAA.
 63. The multispecific polypeptide construct of claim 6, wherein:the at least one antigen binding domain is positioned amino-terminallyrelative to the Fc region and/or is positioned carboxy-terminallyrelative to the CD3 binding region of one of the first or secondpolypeptide of the multispecific polypeptide construct; or the at leastone antigen binding domain comprises a first and a second antigenbinding domain and the first antigen binding domain is positionedamino-terminally relative to the Fc region of the multispecificpolypeptide construct and the second antigen binding domain ispositioned carboxy-terminally relative to the CD3 binding region of themultispecific polypeptide construct.
 64. (canceled)
 65. Themultispecific polypeptide construct of claim 1, wherein the antigenbinding domain, or independently each of the antigen binding domains:comprises an extracellular domain or binding fragment thereof of thenative cognate binding partner of the TAA, or a variant thereof thatexhibits binding activity to the TAA; or is an antibody orantigen-binding fragment thereof selected from the group consisting of aFab fragment, a F(ab′)2 fragment, an Fv fragment, a scFv, a scAb, a dAb,a single domain heavy chain antibody, and a single domain light chainantibody.
 66. (canceled)
 67. The multispecific polypeptide construct ofclaim 65, wherein the antibody or antigen-binding fragment thereof is aFv, a scFv, a Fab, or a single domain antibody (sdAb). 68-77. (canceled)78. The multispecific polypeptide construct of claim 6, wherein theantigen binding domain, or independently each of the antigen bindingdomains, binds to a tumor antigen selected from among 1-92-LFA-3, 5T4,Alpha-4 integrin, Alpha-V integrin, alpha4beta1 integrin, alpha4beta7integrin, AGR2, Anti-Lewis-Y, Apelin J receptor, APRIL, B7-H3, B7-H4,BAFF, BTLA, C5 complement, C-242, CA9, CA19-9, (Lewis a), Carbonicanhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25,CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47,CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD117,CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138, CD166, CD172A,CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), CLAUDIN-3, CLAUDIN-4,cMet, Collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13,CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1, EDA,EDB, EGFR, EGFRviii, Endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2,EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3,FGFR4, FLT-3, Folate receptor alpha (FRα), GAL3ST1, G-CSF, G-CSFR, GD2,GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GP IIb/IIIa receptors, Gp130,GPIIB/IIIA, GPNMB, GRP78, HER2/neu, HER3, HER4, HGF, hGH, HVEM,Hyaluronidase, ICOS, IFNalpha, IFNbeta, IFNgamma, IgE, IgE Receptor(FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R,IL-12Rbeta1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R,IL27/IL27R (wsx1), IL29, IL-31R, IL31/IL31R, IL2R, IL4, IL4R, IL6, IL6R,Insulin Receptor, Jagged Ligands, Jagged 1, Jagged 2, KISS1-R, LAG-3,LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, Mesothelin,MRP4, MUC1, Mucin-16 (MUC16, CA-125), Na/K ATPase, NGF, Nicastrin, NotchReceptors, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, OX-40, PAR2,PDGF-AA, PDGF-BB, PDGFRalpha, PDGFRbeta, PD-1, PD-L1, PD-L2,Phosphatidyl-serine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4,Sphingosine 1 Phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFbeta,TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFalpha,TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, Transferrin, Transferrin receptor,TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D,VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3.
 79. Themultispecific polypeptide construct of any of claim 6, wherein themultispecific polypeptide construct comprises: at least a first antigenbinding domain and a second antigen binding domain, wherein the firstantigen binding domain and second antigen binding domain bind to thesame TAA; at least a first antigen binding domain and a second antigenbinding domain wherein the first antigen binding domain and the secondantigen binding domain binds different epitopes of the same TAA; atleast a first antigen binding domain and a second antigen binding domainwherein the first antigen binding domain and the second antigen bindingdomain bind the same epitope of the same TAA; or at least a firstantigen binding domain and a second antigen binding domain wherein thefirst antigen binding domain and the second antigen binding domain binda different TAA. 80-86. (canceled)
 87. The multispecific polypeptideconstruct of claim 6, wherein the Fv antibody fragment comprises adisulfide stabilized anti-CD3 binding Fv fragment (dsFv). 88-91.(canceled)
 92. The multispecific polypeptide construct of claim 6,wherein the multispecific polypeptide construct is conjugated to anagent selected from among the following: a therapeutic agent, anantineoplastic agent, a toxin or fragment thereof, a detectable moiety,and a diagnostic agent.
 93. (canceled)
 94. (canceled)
 95. Apolynucleotide(s) encoding the mutispecific polypeptide constructs ofclaim
 6. 96-101. (canceled)
 102. A vector, comprising the polynucleotideof claim
 95. 103. (canceled)
 104. (canceled)
 105. A cell, comprising thepolynucleotide or polynucleotides of claim
 95. 106-108. (canceled) 109.A method of producing a multispecific polypeptide construct, the methodcomprising introducing into a cell the polynucleotide or polynucleotidesof claim 95 and culturing the cell under conditions to produce themultispecific polypeptide construct. 110-113. (canceled)
 114. Apharmaceutical composition comprising the multispecific polypeptideconstruct of claim 6 and a pharmaceutically acceptable carrier. 115.(canceled)
 116. A method of stimulating or inducing an immune response,the method comprising contacting a target cell and a T cell with themultispecific polypeptide construct of claim 6, said target cellexpressing a tumor associated antigen recognized by the multispecificpolypeptide construct. 117-125. (canceled)
 126. A method of treating adisease or condition in a subject, the method comprising administering,to a subject in need thereof, a therapeutically effective amount of themultispecific polypeptide construct of claim
 6. 127. The method of claim126, wherein the disease or condition is a tumor or a cancer. 128.(canceled)
 129. The multispecific polypeptide construct of claim 1,wherein the Fv antibody fragment comprises a disulfide stabilizedanti-CD3 binding Fv fragment (dsFv).
 130. The multispecific polypeptideconstruct of claim 5, wherein the antigen binding domain, orindependently each of the antigen binding domains: comprises anextracellular domain or binding fragment thereof of the native cognatebinding partner of the TAA, or a variant thereof that exhibits bindingactivity to the TAA; or is an antibody or antigen-binding fragmentthereof selected from the group consisting of a Fab fragment, a F(ab′)2fragment, an Fv fragment, a scFv, a scAb, a dAb, a single domain heavychain antibody, and a single domain light chain antibody.
 131. Themultispecific polypeptide construct of claim 1, wherein thenon-cleavable linker is or comprises an amino acid sequence selectedfrom among GGS; GGGGS (SEQ ID NO: 149); GGGGGS (SEQ ID NO: 135); (GGS)2(SEQ ID NO: 10); GGSGGSGGS (SEQ ID NO: 11); GGSGGSGGSGGS (SEQ ID NO:12); GGSGGSGGSGGSGGS (SEQ ID NO: 13); GGGGGSGGGGGSGGGGGS (SEQ ID NO:119); GGSGGGGSGGGGSGGGGS (SEQ ID NO: 147); and GGGGSGGGGSGGGGS (SEQ IDNO:170).
 132. The multispecific polypeptide construct of claim 5,wherein the non-cleavable linker is or comprises an amino acid sequenceselected from among GGS; GGGGS (SEQ ID NO: 149); GGGGGS (SEQ ID NO:135); (GGS)2 (SEQ ID NO: 10); GGSGGSGGS (SEQ ID NO: 11); GGSGGSGGSGGS(SEQ ID NO: 12); GGSGGSGGSGGSGGS (SEQ ID NO: 13); GGGGGSGGGGGSGGGGGS(SEQ ID NO: 119); GGSGGGGSGGGGSGGGGS (SEQ ID NO: 147); andGGGGSGGGGSGGGGS (SEQ ID NO:170).
 133. A polynucleotide(s) encoding themutispecific polypeptide constructs of claim
 1. 134. A cell, comprisingthe polynucleotide or polynucleotides of claim
 133. 135. A method ofproducing a multispecific polypeptide construct, the method comprisingintroducing into a cell the polynucleotide or polynucleotides of claim133 and culturing the cell under conditions to produce the multispecificpolypeptide construct.
 136. A pharmaceutical composition comprising themultispecific polypeptide construct of claim 1 and a pharmaceuticallyacceptable carrier.
 137. A method of stimulating or inducing an immuneresponse, the method comprising contacting a target cell and a T cellwith the multispecific polypeptide construct of claim 1, said targetcell expressing a tumor associated antigen recognized by themultispecific polypeptide construct.
 138. A method of treating a diseaseor condition in a subject, the method comprising administering, to asubject in need thereof, a therapeutically effective amount of themultispecific polypeptide construct-of claim
 1. 139. A polynucleotide(s)encoding the mutispecific polypeptide constructs of claim
 5. 140. Acell, comprising the polynucleotide or polynucleotides of claim 139.141. A method of producing a multispecific polypeptide construct, themethod comprising introducing into a cell the polynucleotide orpolynucleotides of claim 139 and culturing the cell under conditions toproduce the multispecific polypeptide construct.
 142. A pharmaceuticalcomposition comprising the multispecific polypeptide construct of claim5 and a pharmaceutically acceptable carrier.
 143. A method ofstimulating or inducing an immune response, the method comprisingcontacting a target cell and a T cell with the multispecific polypeptideconstruct of claim 5, said target cell expressing a tumor associatedantigen recognized by the multispecific polypeptide construct.
 144. Amethod of treating a disease or condition in a subject, the methodcomprising administering, to a subject in need thereof, atherapeutically effective amount of the multispecific polypeptideconstruct-of claim 5.